Compositions and methods for use for antibodies against sclerostin

ABSTRACT

The present invention relates to antibodies against sclerostin and compositions and methods of use for said antibodies to treat a pathological disorder that is mediated by sclerostin or disease related to bone abnormalities such as osteoporosis.

This application is a continuation of U.S. patent application Ser. No.12/249,050 filed on Oct. 10, 2008, which issued as U.S. Pat. No.7,879,322 on Feb. 1, 2011, and which claims benefit of EP ApplicationNo. 07118414.7 filed on Oct. 12, 2007, EP Application No. 08151911.8filed on Feb. 25, 2008, and EP Application No. 08161342.4 filed on Jul.29, 2008, the entire disclosures of which are hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates to antibodies against sclerostin andcompositions and methods of use for said antibodies to treat apathological disorder that is mediated by sclerostin or disease relatedto bone abnormalities such as osteoporosis.

BACKGROUND OF THE INVENTION

The SOST gene encodes the protein sclerostin which is a 213 amino acidssecreted glycoprotein. Sclerostin is a member of the super-family ofcystine-knot containing factors. Sclerostin is related to theDAN/Cerberus protein family, which interferes directly with BMPsignaling by inhibiting the binding of BMP to the receptors and thus theBMP signaling cascade (Avsian-Kretchmer, Mol Endocrinol 2004,18(1):1-12).

Sclerostin mRNA expression is detected in adult humans predominantly inbone and kidney. Sclerostin protein is detectable predominantly in bone.Within bone its expression is restricted to the mature and terminallydifferentiated bone forming cells, the osteocytes.

Sclerostin is a potent negative regulator of bone formation in men andmice. Lack of SOST expression gives rise to sclerosteosis (Balemans etal. Hum Mol Genet., 2001, 10(5):537-43; Brunkow et al. Am J Hum Genet,2001, 68(3):577-89). Patients suffer from life-long bone overgrowthresulting in increased bone mineral density and strength. They displayno other endocrinological abnormalities—all complications theyexperience during their life-time are related to the abnormalaccumulation of bone. Heterozygous carriers for this recessive disorderalso display increased bone mass (Gardner et al. J Clin EndocrinolMetab, 2005, 90(12):6392-5). This phenotype can be recapitulated in SOSTdeficient mice and its overexpression results in osteopenia. FurthermoreVan Buchem disease [MIM 239100]—a phenotypic copy of sclerosteosis—iscaused by SOST misregulation due to the genomic deletion of a long-rangebone enhancer (Balemans et al. J Med Gene, 2002, 39(2):91-7; Loots etal., Genome Res, 2005, 15(7):928-35). Finally, SOST is down-regulated byparathyroid hormone—a clinically validated bone forming principle—duringbone formation suggesting that part of the anabolic action of PTH mightbe mediated via SOST (Keller and Kneissel Bone, 2005, 37(2):148-58).

Sclerostin binds BMPs (bone morphogenic proteins) and can act as a BMPantagonist in vitro (Winkler et al. EMBO J., 2003, 22(23):6267-76).Sclerostin also acts as a negative regulator of canonical Wnt signaling,either directly by binding to LRP5/LRP6 (Li et al. J Biol Chem., 2005,20; 280(20); Semenov, J Biol Chem. 2006 Oct. 19; van Bezooijen et al. JBone Miner Res, 2006, Oct. 10), or indirectly (Winkler et al. J BiolChem., 2005, 28; 280(4):2498-502).

Lack of sclerostin expression results in high bone formation, while boneresorption is undisturbed (Sclerosteosis, Van Buchem disease) (Balemanset al. 2001; Brunkow et al. Am J Hum Genet, 2001, 68(3):577-89, Balemanset al. 2006; Loots et al., Genome Res, 2005, 15(7):928-35).

Few of the presently available treatments for skeletal disorders canincrease the bone density of adults, and most of the presently availabletreatments work primarily by inhibiting further bone resorption ratherthan stimulating new bone formation.

One example of a medicament used for treating bone loss is estrogen.However, it is not clear whether or not estrogen has any beneficial longterm effects. Furthermore, estrogen may carry the risk of increasing theprevalence of various types of tumors, such as breast and endometrialcancer. Other current therapeutic approaches to osteoporosis includebisphosphonates (e.g., Fosamax™, Actonel™, Bonviva™, Zometa™,olpadronate, neridronate, skelid, bonefos), parathyroid hormone,calcilytics, calcimimetics (e.g., cinacalcet), statins, anabolicsteroids, lanthanum and strontium salts, and sodium fluoride. Suchtherapeutics, however, are often associated with undesirable sideeffects.

SUMMARY OF THE INVENTION

An embodiment of the invention herein provides an antibody or afunctional protein comprising an antigen-binding portion of saidantibody for a target in sclerostin polypeptide (SEQ ID NO:155),characterized in that the antibody or functional protein specificallybinds to sclerostin polypeptide and can increase at least one of boneformation, bone mineral density, bone mineral content, bone mass, bonequality and bone strength in a mammal.

In one embodiment, the antibodies according to the invention have theability to reverse sclerostin inhibition of in vitro bonemineralization. In a related embodiment, they have the ability toreverse sclerostin inhibition of wnt-1 mediated signaling pathway. Inanother related embodiment, they disrupt sclerostin LRP6 binding and canblock the inhibitory effect that sclerostin has at high doses on BMPinduced Smad1 phosphorylation. In another embodiment, the antibodies ofthe invention bind to a region of sclerostin between amino acids 112 and126 inclusive (i.e. said region consists of amino acids 112 to 126 ofSEQ ID NO:155) of SEQ ID NO:155 and/or the region between amino acids160-174 inclusive (i.e. said region consists of amino acids 160 to 174of SEQ ID NO:155) of SEQ ID NO:155, and more specifically, bind to aregion comprising both ARLLPNAIGRGKWWR (SEQ ID NO 156) andRLVASCKCKRLTRFH (SEQ ID NO 157).

Sclerostin inhibits wnt1-mediated activation of STF (Supertopflash,reporter readout for canonical wnt signaling) in HEK293 cells. In someembodiments, the antibodies of the invention restore the wnt signalingreporter readout in a highly reproducible manner.

The observed inhibitory effect of the antibodies according to theinvention on sclerostin action in the Wnt signaling reporter assay innon-osteoblastic cells has been shown to translate into induction ofbone formation responses due to sclerostin inhibition in viva Indeed, invivo experiments in aged rodents show that the antibodies according tothe invention promotes strong bone anabolism. The bone mass increasereached the effect level of daily intermittent treatment with extremelyhigh anabolic doses of parathyroid hormone (which was used as a positivecontrol).

Therefore, according to another preferred embodiment, the antibodiesaccording to the invention have affinities to sclerostin in the low pMrange and inhibit sclerostin impact on wnt signalling with an IC₅₀around 10 nM.

More preferably, in another preferred embodiment, the antibodiesaccording to the invention bind to a region of sclerostin comprisedbetween amino acids 112 and 126 inclusive (i.e. said region consists ofamino acids 112 to 126 of SEQ ID NO:155) and between amino acids 160 and174 inclusive (i.e. said region consists of amino acids 160 to 174 ofSEQ ID NO:155) of SEQ ID NO:155, and more specifically a region thatoverlaps at least the following peptides ARLLPNAIGRGKWWR (SEQ ID NO:156) and RLVASCKCKRLTRFH (SEQ ID NO:157), respectively, and haveaffinities to sclerostin in the low pM range and inhibit sclerostinimpact on wnt signalling with an IC₅₀ around 10 nM. Such antibodies havethe capacity to increase bone mass in the axial and appendicularskeleton of mouse animal model at the effect level of daily subcutaneoustreatment with an extremely high anabolic dose of parathyroid hormone(positive control) and are therefore useful in the treatment of diseaserelated to bone abnormalities such as osteoporosis.

Further embodiments include compositions comprising the antibodies ofthe invention in combination with alternative therapies for treatingosteoporosis, such as bisphosphonates, parathyroid hormone, parathyroidhormone releasing agents (calcilytics), LRP4 neutralising antibodies andDKK-1 neutralising antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Effect of MOR05813_IgG2lambda in the wnt-1 assay

FIG. 2: MOR05813_IgG2lambda in BMP-2-induced mineralization in MC3T3-1bcells

FIG. 3: Effect of MOR05813_IgG2lambda in the LRP6-SOST ELISA

FIG. 4: Effect of MOR05813_gG2lambda in the Phospho-Smad1 assay

FIG. 5: A—Effect of LRP4 knockdown (siRNA) on SOST inhibitory action inthe wnt-1 assay in Hek293 cells (Black numbers: relative to STFactivities in the absence of SOST, Bold black numbers: ratio of STFactivities in the presence/absence of SOST); B—Specificity of the effectof LRP4 overexpression on SOST IC₅₀ and Dkk1 IC50 in the wnt-1 assay inHek293 cells; C—Specificity of the effect of LRP4 overexpression on SOSTand Dkk1 inhibitory action in the wnt-1 assay C28a2 cells; D—Specificityof the effect of LRP4 knockdown (siRNA) on SOST and Dkk1 inhibitoryaction in the wnt-1 assay in Hek293 cells; E-Modulation of the activityof MOR05813 by LRP4

FIG. 6: Mouse study, in vivo pQCT—2.5 weeks treatment with MOR05813increases total bone mineral content in the proximal tibia metaphysis

FIG. 7: Mouse study, in vivo pQCT—2.5 weeks treatment with MOR05813increases total bone mineral density in the proximal tibia metaphysis

FIG. 8: Mouse study, in vivo pQCT—2.5 weeks treatment with MOR05813increases cortical thickness in the proximal tibia metaphysis

FIG. 9: Mouse study, in vivo uQCT—2.5 weeks treatment with MOR05813increases cancellous bone volume in the proximal tibia metaphysis

FIG. 10: Mouse study, in vivo uQCT—2.5 weeks treatment with MOR05813increases trabecular thickness in the proximal tibia metaphysis

FIG. 11: Mouse study, in vivo pQCT—5 weeks treatment with MOR05813increases total bone mineral density further in the proximal tibiametaphysis

FIG. 12: Mouse study, ex vivo DEXA—5 weeks treatment with MOR05813increases bone mineral density further in the tibia

FIG. 13: Mouse study, ex vivo DEXA—5 weeks treatment with MOR05813increases bone mineral density further in the femur

FIG. 14: Mouse study, ex vivo DEXA—5 weeks treatment with MOR05813increases bone mineral density further in the spine

FIG. 15: Mouse study, ex vivo histomorphometry—2.5 weeks treatment withMOR05813 increases bone formation rates in the appendicular skeleton(distal femur metaphysis)

FIG. 16: Mouse study, ex vivo histomorphometry—2.5 weeks treatment withMOR05813 increases mineral apposition rate in the appendicular skeleton(distal femur metaphysis)

FIG. 17: Mouse study, ex vivo histomorphometry—2.5 weeks treatment withMOR05813 increases mineralizing surface in the appendicular skeleton(distal femur metaphysis)

FIG. 18: Mouse study, ex vivo histomorphometry—2.5 weeks treatment withMOR05813 increases bone formation rates in the axial skeleton (lumbarvertebra)

FIG. 19: Mouse study, ex vivo histomorphometry—2.5 weeks treatment withMOR05813 does not affect bone resorption in the appendicular skeleton(distal femur metaphysis), as measured by osteoclast surface

FIG. 20: ELISA showing effect of MOR05813_IgG2lambda on SOST binding ofLRP6. 0.9 nM SOST was used in each case

FIG. 21: Mouse study, in vivo pQCT following co-treatment with MOR05813and zoledronic acid, (A) Total bone mineral density, (B) Total bonemineral content, (C) Cortical thickness, and (D) Cancellous bone mineraldensity

FIG. 22: Mouse study, in vivo pQCT: treatment with MOR05813 followingalendronate (alen) pre-treatment, (A) Total bone mineral density, (B)Total bone mineral content, (C) Cortical thickness, and (D) Cancellousbone mineral density

FIG. 23: Mouse study, in vivo pQCT following anabolic co-treatment withMOR05813 and (i) anti-DKK1, or (ii) PTH, (A) Total bone mineral density,(B) Total bone mineral content, (C) Cortical thickness, and (D)Cancellous bone mineral density

DETAILED DESCRIPTION

The present invention relates to isolated antibodies, particularly humanantibodies, that bind specifically to sclerostin and that inhibitfunctional properties of sclerostin. In certain embodiments, theantibodies of the invention are derived from particular heavy and lightchain sequences and/or comprise particular structural features such asCDR regions comprising particular amino acid sequences. The inventionprovides isolated antibodies, methods of making such antibodies,immunoconjugates and multivalent or multispecific molecules comprisingsuch antibodies and pharmaceutical compositions containing theantibodies, immunoconjugates or bispecific molecules of the invention.The invention also relates to methods of using the antibodies to inhibita disorder or condition associated with the presence of sclerostinexpression, for example, in the treatment a pathological disorder thatis mediated by sclerostin or that is associated with an increased levelof sclerostin; for example, a bone related disease such as osteoporosis.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description. The term “comprising” encompasses“including” as well as “consisting” e.g. a composition “comprising” Xmay consist exclusively of X or may include something additional e.g.X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

The term sclerostin refers to human sclerostin as defined in SEQ ID NO:155. Recombinant human sclerostin can be obtained from R&D Systems(Minneapolis, Minn., USA; 2006 cat #1406-ST-025). Additionally,recombinant mouse sclerostin/SOST is commercially available from R&DSystems (Minneapolis, Minn., USA; 2006 cat #1589-ST-025). U.S. Pat. Nos.6,395,511 and 6,803,453, and U.S. Patent Publications 20040009535 and20050106683 refer to anti-sclerostin antibodies in general.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. A naturally occurring “antibody” is a glycoproteincomprising at least two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as VH) and a heavy chainconstant region. The heavy chain constant region is comprised of threedomains, CH1, CH2 and CH3. Each light chain is comprised of a lightchain variable region (abbreviated herein as VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. The VH and VL regions can be further subdivided into regionsof hypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRsarranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavyand light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antigenportion”), as used herein, refers to full length or one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., sclerostin). It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and CH1 domains; a F(ab)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment consisting of the V_(H) and CH1 domains; a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), whichconsists of a VH domain; and an isolated complementarity determiningregion (CDR).

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc.Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding region” ofan antibody. These antibody fragments are obtained using conventionaltechniques known to those of skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

An “isolated antibody”, as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically bindssclerostin is substantially free of antibodies that specifically bindantigens other than sclerostin). An isolated antibody that specificallybinds sclerostin may, however, have cross-reactivity to other antigens,such as sclerostin molecules from other species. Moreover, an isolatedantibody may be substantially free of other cellular material and/orchemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutatedversions of human germline sequences or antibody containing consensusframework sequences derived from human framework sequences analysis asdescribed in Knappik, et al. (2000. J Mol Biol 296, 57-86).

The human antibodies of the invention may include amino acid residuesnot encoded by human sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic nonhuman animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM, IgE,IgG such as IgG1 or IgG2) that is provided by the heavy chain constantregion genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen”.

As used herein, an antibody that “specifically binds to sclerostinpolypeptide” is intended to refer to an antibody that binds tosclerostin polypeptide with a K_(D) of 1×10⁻⁸ M or less, 1×10⁻⁹ M orless, or 1×10⁻¹⁰ M or less. An antibody that “cross-reacts with anantigen other than sclerostin” is intended to refer to an antibody thatbinds that antigen with a K_(D) of 0.5×10⁻⁸ M or less, 5×10⁻⁹ M or less,or 2×10⁻⁹ M or less. An antibody that “does not cross-react with aparticular antigen” is intended to refer to an antibody that binds tothat antigen, with a K_(D) of 1.5×10⁻⁸ M or greater, or a K_(D) ofbetween 5×10⁻⁸ M and 10×10⁻⁸ M, or 1×10⁻⁷ M or greater. In certainembodiments, such antibodies that do not cross-react with the antigenexhibit essentially undetectable binding against these proteins instandard binding assays.

As used herein, an antibody that “blocks the inhibitory effect ofsclerostin in a cell based wnt signaling assay” is intended to refer toan antibody that restores wnt induced signaling in the presence ofsclerostin in a cell-based super top flash (STF) assay with an IC₅₀ lessthan 1 mM, 100 nM, 20 nM, 10 nM or less. Such STF assay is described inmore details in the examples below.

As used herein, an antibody that “blocks the inhibitory effect ofsclerostin in a cell based mineralization assay” is intended to refer toan antibody that restores BMP2 induced mineralisation in the presence ofsclerostin in a cell-based assay with an IC₅₀ less than 1 mM, 500 nM,100 nM, 10 nM, 1 nM or less. Such assay is described in more details inthe examples below.

As used herein, an antibody that “blocks the inhibitory effect ofsclerostin in Smad1 phosphorylation assay” is intended to refer to anantibody that restores BMP6 induced Smad1 phosphorylation in thepresence of sclerostin in a cell based assay with an IC₅₀ less than 1mM, 500 nM, 100 nM, 10 nM, 1 nM or less. Such assay is described in moredetails in the examples below.

As used herein, an antibody that “inhibits binding of sclerostin to theLRP-6” refers to an antibody that inhibits sclerostin binding to LRP-6with a IC₅₀ of 1 mM, 500 nM, 100 nM, 10 nM, 5 nM, 3 nM, 1 nM or less.Such assay is described in more details in the examples below.

As used herein, an antibody that “increases bone formation and mass anddensity” refers to an antibody that is capable of reaching boneformation, mass and density at the level of daily intermittent treatmentwith high anabolic dose of PTH as shown in the Example 10.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(D),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A method for determining the K_(D) of anantibody is by using surface plasmon resonance, or using a biosensorsystem such as a Biacore® system.

As used herein, the term “Affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with antigen at numeroussites; the more interactions, the stronger the affinity.

As used herein, the term “Avidity” refers to an informative measure ofthe overall stability or strength of the antibody-antigen complex. It iscontrolled by three major factors: antibody epitope affinity; thevalence of both the antigen and antibody; and the structural arrangementof the interacting parts. Ultimately these factors define thespecificity of the antibody, that is, the likelihood that the particularantibody is binding to a precise antigen epitope.

In order to get a higher avidity probe, a dimeric conjugate can beconstructed, thus making low affinity interactions (such as with thegermline antibody) more readily detected by FACS. In addition, anothermeans to increase the avidity of antigen binding involves generatingdimmers, trimers or multimers of any of the constructs described hereinof the anti-sclerostin antibodies. Such multimers may be generatedthrough covalent binding between individual modules, for example, byimitating the natural C-to-N-terminus binding or by imitating antibodydimers that are held together through their constant regions. The bondsengineered into the Fc/Fc interface may be covalent or non-covalent. Inaddition, dimerizing or multimerizing partners other than Fc can be usedin sclerostin hybrids to create such higher order structures. Forexample, it is possible to use multimerizing domains such as trimerizingdomain described in Borean (WO2004039841) or pentamerizing domaindescribed in published patent application WO98/18943.

As used herein, the term “cross-reactivity” refers to an antibody orpopulation of antibodies binding to epitopes on other antigens. This canbe caused either by low avidity or specificity of the antibody or bymultiple distinct antigens having identical or very similar epitopes.Cross reactivity is sometimes desirable when one wants general bindingto a related group of antigens or when attempting cross-species labelingwhen the antigen epitope sequence is not highly conserved in evolution.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, 10⁻⁹ M or less, or 10⁻¹⁰ Mor less for a target antigen. However, “high affinity” binding can varyfor other antibody isotypes. For example, “high affinity” binding for anIgM isotype refers to an antibody having a K_(D) of 10⁻⁷ M or less, or10⁻⁸ M or less.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows chickens, amphibians, reptiles, etc.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia or Trichoderma, a Chinese HamsterOvary cell (CHO) or a human cell. The optimized nucleotide sequence isengineered to retain completely or as much as possible the amino acidsequence originally encoded by the starting nucleotide sequence, whichis also known as the “parental” sequence. The optimized sequences hereinhave been engineered to have codons that are preferred in mammaliancells, however optimized expression of these sequences in othereukaryotic cells is also envisioned herein. The amino acid sequencesencoded by optimized nucleotide sequences are also referred to asoptimized.

Various aspects of the invention are described in further detail in thefollowing subsections.

Standard assays to evaluate the binding ability of the antibodies towardsclerostin of various species are known in the art, including forexample, ELISAs, western blots and RIAs. Suitable assays are describedin detail in the Examples. The binding kinetics (e.g., binding affinity)of the antibodies also can be assessed by standard assays known in theart, such as by Biacore analysis. Assays to evaluate the effects of theantibodies on functional properties of sclerostin (e.g., receptorbinding, preventing or ameliorating osteolysis) are described in furtherdetail in the Examples.

Accordingly, an antibody that “inhibits” one or more of these sclerostinfunctional properties (e.g., biochemical, immunochemical, cellular,physiological or other biological activities, or the like) as determinedaccording to methodologies known to the art and described herein, willbe understood to relate to a statistically significant decrease in theparticular activity relative to that seen in the absence of the antibody(or when a control antibody of irrelevant specificity is present). Anantibody that inhibits sclerostin activity effects such a statisticallysignificant decrease by at least 10% of the measured parameter, by atleast 50%, 80% or 90%, and in certain embodiments an antibody of theinvention may inhibit greater than 95%, 98% or 99% of sclerostinfunctional activity.

The terms “cross-block”, “cross-blocked” and “cross-blocking” are usedinterchangeably herein to mean the ability of an antibody or otherbinding agent to interfere with the binding of other antibodies orbinding agents to sclerostin in a standard competitive binding assay.

The ability or extent to which an antibody or other binding agent isable to interfere with the binding of another antibody or bindingmolecule to sclerostin, and therefore whether it can be said tocross-block according to the invention, can be determined using standardcompetition binding assays. One suitable assay involves the use of theBiacore technology (e.g. by using the BIAcore 3000 instrument (Biacore,Uppsala, Sweden)), which can measure the extent of interactions usingsurface plasmon resonance technology. Another assay for measuringcross-blocking uses an ELISA-based approach.

Further details on both methods are given in the Examples.

According to the invention, a cross-blocking antibody or other bindingagent according to the invention binds to sclerostin in the describedBIAcore cross-blocking assay such that the recorded binding of thecombination (mixture) of the antibodies or binding agents is between 80%and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding,specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximumtheoretical binding, and more specifically between 70% and 0.1% (e.g.70% to 4%), and more specifically between 65% and 0.1% (e.g. 65% to 4%)of maximum theoretical binding (as defined above) of the two antibodiesor binding agents in combination.

An antibody is defined as cross-blocking in the ELISA assay as describedin the Examples, if the solution phase anti-sclerostin antibody is ableto cause a reduction of between 60% and 100%, specifically between 70%and 100%, and more specifically between 80% and 100%, of the sclerostindetection signal (i.e. the amount of sclerostin bound by the coatedantibody) as compared to the sclerostin detection signal obtained in theabsence of the solution phase anti-sclerostin antibody (i.e. thepositive control wells).

Monoclonal Antibodies

Antibodies of the invention include the human monoclonal antibodies,isolated as described, in the Examples. The V_(H) amino acid sequencesof isolated antibodies of the invention are shown in SEQ ID NOs: 69-77.The V_(L) amino acid sequences of isolated antibodies of the inventionare shown in SEQ ID NOs: 80-88 respectively. The corresponding preferredfull length heavy chain amino acid sequences of antibodies of theinvention are shown in SEQ ID NO:113-121. The corresponding preferredfull length light chain amino acid sequences of antibodies of theinvention are shown in SEQ ID NO:124-132 respectively. Other antibodiesof the invention include amino acids that have been mutated, yet have atleast 60, 70, 80, 90 or 95 percent or more identity in the CDR regionswith the CDR regions depicted in the sequences described above. In someembodiments, the invention includes mutant amino acid sequences whereinno more than 1, 2, 3, 4 or 5 amino acids have been mutated in the CDRregions when compared with the CDR regions depicted in the sequencedescribed above.

Further, variable heavy chain parental nucleotide sequences are shown inSEQ ID NOs 89-90. Variable heavy chain parental nucleotide sequences areshown in SEQ ID NOs 100-101. Full length light chain nucleotidesequences optimized for expression in a mammalian cell are shown in SEQID NOs 146-154. Full length heavy chain nucleotide sequences optimizedfor expression in a mammalian cell are shown in SEQ ID NOs 135-143. Fulllength light chain amino acid sequences encoded by optimized light chainnucleotide sequences are shown in SEQ ID NOs 124-132. Full length heavychain amino acid sequences encoded by optimized heavy chain nucleotidesequences are shown in SEQ ID NOs 113-121. Other antibodies of theinvention include amino acids or nucleic acids that have been mutated,yet have at least 60, 70, 80, 90 or 95 percent or more identity to thesequences described above. In some embodiments, the invention includesmutant amino acid sequences wherein no more than 1, 2, 3, 4 or 5 aminoacids have been mutated in the variable regions when compared with thevariable regions depicted in the sequence described above, whileretaining substantially the same therapeutic activity.

Since each of these antibodies can bind to sclerostin, the V_(H), V_(L),full length light chain, and full length heavy chain sequences(nucleotide sequences and amino acid sequences) can be “mixed andmatched” to create other anti-sclerostin binding molecules of theinvention. Sclerostin binding of such “mixed and matched” antibodies canbe tested using the binding assays described above and in the Examples(e.g., ELISAs). When these chains are mixed and matched, a V_(H)sequence from a particular V_(H)/V_(L) pairing should be replaced with astructurally similar V_(H) sequence. Likewise a full length heavy chainsequence from a particular full length heavy chain/full length lightchain pairing should be replaced with a structurally similar full lengthheavy chain sequence. Likewise, a V_(L) sequence from a particularV_(H)/V_(L) pairing should be replaced with a structurally similar V_(L)sequence. Likewise a full length light chain sequence from a particularfull length heavy chain/full length light chain pairing should bereplaced with a structurally similar full length light chain sequence.Accordingly, in one aspect, the invention provides an isolatedmonoclonal antibody or antigen binding region thereof having: a heavychain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 69-77; and a light chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 80-88; wherein the antibody specifically bindsto sclerostin.

In another aspect, the invention provides:

(i) an isolated monoclonal antibody having: a full length heavy chaincomprising an amino acid sequence that has been optimized for expressionin the cell of a mammal selected from the group consisting of SEQ IDNOs:113-121; and a full length light chain comprising an amino acidsequence that has been optimized for expression in the cell of a mammalselected from the group consisting of SEQ ID NOs:124-132; or

(ii) a functional protein comprising an antigen binding portion thereof.

In another aspect, the invention provides:

(i) an isolated monoclonal antibody having: a full length heavy chaincomprising a nucleotide sequence that has been optimized for expressionin the cell of a mammal selected from the group consisting of SEQ IDNOs:135-143; and a full length light chain comprising a nucleotidesequence that has been optimized for expression in the cell of a mammalselected from the group consisting of SEQ ID NOs:146-154; or,

(ii) a functional protein comprising an antigen binding portion thereof.

In yet another aspect, the invention provides antibodies that comprisethe heavy chain and light chain CDR1s, CDR2s and CDR3s of theantibodies, or combinations thereof. The amino acid sequences of theV_(H) CDR1s of the antibodies are shown in SEQ ID NOs: 1-11. The aminoacid sequences of the V_(H) CDR2s of the antibodies are shown in SEQ IDNOs: 12-22. The amino acid sequences of the V_(H) CDR3s of theantibodies are shown in SEQ ID NOs: 23-33. The amino acid sequences ofthe V_(L) CDR1s of the antibodies are shown in SEQ ID NOs: 34-44. Theamino acid sequences of the V_(L) CDR2s of the antibodies are shown inSEQ ID NOs: 45-55. The amino acid sequences of the V_(L) CDR3s of theantibodies are shown in SEQ ID NOs: 56-66. The CDR regions aredelineated using the Kabat system (Kabat, E. A., et al., 1991 Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242).

Given that each of these antibodies can bind to sclerostin and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(L) CDR1, 2 and 3sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and matched), although each antibody mustcontain a V_(H) CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3 to create otheranti-sclerostin binding molecules of the invention. Sclerostin bindingof such “mixed and matched” antibodies can be tested using the bindingassays described above and in the Examples (e.g., ELISAs). When V_(H)CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequencefrom a particular V_(H) sequence should be replaced with a structurallysimilar CDR sequence(s). Likewise, when V_(L) CDR sequences are mixedand matched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(L)sequence should be replaced with a structurally similar CDR sequence(s).It will be readily apparent to the ordinarily skilled artisan that novelV_(H) and V_(L) sequences can be created by substituting one or moreV_(H) and/or V_(L) CDR region sequences with structurally similarsequences from the CDR sequences shown herein for monoclonal antibodiesof the present invention.

An isolated monoclonal antibody, or antigen binding region thereof has:a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1-11; a heavy chainvariable region CDR2 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 12-22; a heavy chain variable regionCDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 23-33; a light chain variable region CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 34-44; a light chain variable region CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:45-55; and a light chain variable region CDR3 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 56-66;wherein the antibody specifically binds sclerostin.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 3; a heavy chain variable region CDR2 of SEQID NO: 14; a heavy chain variable region CDR3 of SEQ ID NO: 25; a lightchain variable region CDR1 of SEQ ID NO: 36; a light chain variableregion CDR2 of SEQ ID NO: 47; and a light chain variable region CDR3 ofSEQ ID NO: 58.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 4; a heavy chain variable region CDR2 of SEQID NO: 15; a heavy chain variable region CDR3 of SEQ ID NO: 26; a lightchain variable region CDR1 of SEQ ID NO: 37; a light chain variableregion CDR2 of SEQ ID NO: 48; and a light chain variable region CDR3 ofSEQ ID NO: 59.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 5; a heavy chain variable region CDR2 of SEQID NO: 16; a heavy chain variable region CDR3 of SEQ ID NO: 27; a lightchain variable region CDR1 of SEQ ID NO: 38; a light chain variableregion CDR2 of SEQ ID NO: 49; and a light chain variable region CDR3 ofSEQ ID NO: 60.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 6; a heavy chain variable region CDR2 of SEQID NO: 17; a heavy chain variable region CDR3 of SEQ ID NO: 28; a lightchain variable region CDR1 of SEQ ID NO: 39; a light chain variableregion CDR2 of SEQ ID NO: 50; and a light chain variable region CDR3 ofSEQ ID NO: 61.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 7; a heavy chain variable region CDR2 of SEQID NO: 18; a heavy chain variable region CDR3 of SEQ ID NO: 29; a lightchain variable region CDR1 of SEQ ID NO: 40; a light chain variableregion CDR2 of SEQ ID NO: 51; and a light chain variable region CDR3 ofSEQ ID NO: 62.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 8; a heavy chain variable region CDR2 of SEQID NO: 19; a heavy chain variable region CDR3 of SEQ ID NO: 30; a lightchain variable region CDR1 of SEQ ID NO: 41; a light chain variableregion CDR2 of SEQ ID NO: 52; and a light chain variable region CDR3 ofSEQ ID NO: 63.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 9; a heavy chain variable region CDR2 of SEQID NO: 20; a heavy chain variable region CDR3 of SEQ ID NO: 31; a lightchain variable region CDR1 of SEQ ID NO: 42; a light chain variableregion CDR2 of SEQ ID NO: 53; and a light chain variable region CDR3 ofSEQ ID NO: 64.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 10; a heavy chain variable region CDR2 of SEQID NO: 21; a heavy chain variable region CDR3 of SEQ ID NO: 32; a lightchain variable region CDR1 of SEQ ID NO: 43; a light chain variableregion CDR2 of SEQ ID NO: 54; and a light chain variable region CDR3 ofSEQ ID NO: 65.

In a certain embodiment, the antibody comprises: a heavy chain variableregion CDR1 of SEQ ID NO: 11; a heavy chain variable region CDR2 of SEQID NO: 22; a heavy chain variable region CDR3 of SEQ ID NO: 33; a lightchain variable region CDR1 of SEQ ID NO: 44; a light chain variableregion CDR2 of SEQ ID NO: 55; and a light chain variable region CDR3 ofSEQ ID NO: 66.

As used herein, a human antibody comprises heavy or light chain variableregions or full length heavy or light chains that are “the product of”or “derived from” a particular germline sequence if the variable regionsor full length chains of the antibody are obtained from a system thatuses human germline immunoglobulin genes. Such systems includeimmunizing a transgenic mouse carrying human immunoglobulin genes withthe antigen of interest or screening a human immunoglobulin gene librarydisplayed on phage with the antigen of interest. A human antibody thatis “the product of” or “derived from” a human germline immunoglobulinsequence can be identified as such by comparing the amino acid sequenceof the human antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody. A human antibody that is “the product of” or“derived from” a particular human germline immunoglobulin sequence maycontain amino acid differences as compared to the germline sequence, dueto, for example, naturally occurring somatic mutations or intentionalintroduction of site-directed mutation. However, a selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

In some embodiments, germline immunoglobulin amino acid sequences areselected from those comprising the variable heavy chain sequencesconsisting of SEQ ID NO:67-68 respectively, and the variable light chainsequences consisting of SEQ ID NO:78-79 respectively.

Homologous Antibodies

In yet another embodiment, an antibody of the invention has full lengthheavy and light chain amino acid sequences; full length heavy and lightchain nucleotide sequences, variable region heavy and light chainnucleotide sequences, or variable region heavy and light chain aminoacid sequences that are homologous to the amino acid and nucleotidesequences of the antibodies described herein, and wherein the antibodiesretain the desired functional properties of the anti-sclerostinantibodies of the Invention.

For example, the invention provides an isolated monoclonal antibody (ora functional protein comprising an antigen binding portion thereof)comprising a heavy chain variable region and a light chain variableregion, wherein: the heavy chain variable region comprises an amino acidsequence that is at least 80% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 67-77; the light chainvariable region comprises an amino acid sequence that is at least 80%identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 78-88; the antibody specifically binds to sclerostin, andthe antibody exhibits at least one of the following functionalproperties: the antibody blocks the inhibitory effect of sclerostin in acell based wnt signaling assay, the antibody blocks the inhibitoryeffect of sclerostin in a cell based mineralization assay or blocks theinhibitory effect of sclerostin in Smad1 phosphorylation assay or theantibody inhibits binding of sclerostin to the LRP-6 or the antibodyincreases bone formation and mass and density.

In a further example, the invention provides an isolated monoclonalantibody, (or a functional protein comprising an antigen binding portionthereof) comprising a full length heavy chain and a full length lightchain, wherein: the full length heavy chain comprises an amino acidsequence that is at least 80% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs 111-121; the fulllength light chain comprises an amino acid sequence that is at least 80%identical to an amino acid sequence selected from the group consistingof SEQ ID NOs 122-132; the antibody specifically binds to sclerostin,and the antibody exhibits at least one of the following functionalproperties: the antibody blocks the inhibitory effect of sclerostin in acell based wnt signaling assay, the antibody blocks the inhibitoryeffect of sclerostin in a cell based mineralization assay or blocks theinhibitory effect of sclerostin in Smad1 phosphorylation assay or theantibody inhibits binding of sclerostin to the LRP-6 or the antibodyincreases bone formation and mass and density.

In another example, the invention provides an isolated monoclonalantibody (or a functional protein comprising an antigen binding portionthereof), comprising a full length heavy chain and a full length lightchain, wherein: the full length heavy chain is encoded by a nucleotidesequence that is at least 80% identical to a nucleotide sequenceselected from the group consisting of SEQ ID NOs 133-143; the fulllength light chain comprises a nucleotide sequence that is at least 80%identical to a nucleotide sequence selected from the group consisting ofSEQ ID NOs 144-154; the antibody specifically binds to sclerostin, andthe antibody exhibits at least one of the following functionalproperties: the antibody blocks the inhibitory effect of sclerostin in acell based wnt signaling assay, the antibody blocks the inhibitoryeffect of sclerostin in a cell based mineralization assay or blocks theinhibitory effect of sclerostin in Smad1 phosphorylation assay or theantibody inhibits binding of sclerostin to the LRP-6 or the antibodyincreases bone formation and mass and density.

In various embodiments, the antibody may exhibit one or more, two ormore, or three of the functional properties discussed above. Theantibody can be, for example, a human antibody, a humanized antibody ora chimeric antibody.

In other embodiments, the V_(H) and/or V₁ amino acid sequences may be50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to thesequences set forth above. In other embodiments, the V_(H) and/or V_(L)amino acid sequences may be identical except an amino acid substitutionin no more than 1, 2, 3, 4 or 5 amino acid position(s). An antibodyhaving V_(H) and V_(L) regions having high (i.e., 80% or greater)identity to the V_(H) and V₁ regions of SEQ ID NOs 67-77 and SEQ ID NOs78-88 respectively, can be obtained by mutagenesis (e.g., site-directedor PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ IDNOs: 89-99 and 100-110 respectively, followed by testing of the encodedaltered antibody for retained function (i.e., the functions set forthabove) using the functional assays described herein.

In other embodiments, the full length heavy chain and/or full lengthlight chain amino acid sequences may be 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to the sequences set forth above. Anantibody having a full length heavy chain and full length light chainhaving high (i.e., 80% or greater) identity to the full length heavychains of any of SEQ ID NOs 111-121 and full length light chains of anyof SEQ ID NOs 122-132 respectively, can be obtained by mutagenesis(e.g., site-directed or PCR-mediated mutagenesis) of nucleic acidmolecules encoding SEQ ID NOs 133-143 and SEQ ID NOs 144-154respectively, followed by testing of the encoded altered antibody forretained function (i.e., the functions set forth above) using thefunctional assays described herein.

In other embodiments, the full length heavy chain and/or full lengthlight chain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%,97%, 98% or 99% identical to the sequences set forth above.

In other embodiments, the variable regions of heavy chain and/or lightchain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%or 99% identical to the sequences set forth above

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17, 1988) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al., 1990 J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., 1997 NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http:www.ncbi.nhn.nih.gov.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention has a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences, whereinone or more of these CDR sequences have specified amino acid sequencesbased on the antibodies described herein or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the anti-sclerostin antibodies of the invention.Accordingly, the invention provides an isolated monoclonal antibody, ora functional protein comprising an antigen binding portion thereof,consisting of a heavy chain variable region comprising CDR1, CDR2, andCDR3 sequences and a light chain variable region comprising CDR1, CDR2,and CDR3 sequences, wherein: the heavy chain variable region CDR1 aminoacid sequences are selected from the group consisting of SEQ IDNOs:1-11, and conservative modifications thereof; the heavy chainvariable region CDR2 amino acid sequences are selected from the groupconsisting of SEQ ID NOs: 12-22, and conservative modifications thereof;the heavy chain variable region CDR3 amino acid sequences are selectedfrom the group consisting of SEQ ID NOs: 23-33, and conservativemodifications thereof; the light chain variable regions CDR1 amino acidsequences are selected from the group consisting of SEQ ID NOs: 34-44,and conservative modifications thereof; the light chain variable regionsCDR2 amino acid sequences are selected from the group consisting of SEQID NOs: 45-55, and conservative modifications thereof; the light chainvariable regions of CDR3 amino acid sequences are selected from thegroup consisting of SEQ ID NOs: 56-66, and conservative modificationsthereof; the antibody specifically binds to sclerostin, and the antibodyexhibits at least one of the following functional properties: theantibody blocks the inhibitory effect of sclerostin in a cell based wntsignaling assay, the antibody blocks the inhibitory effect of sclerostinin a cell based mineralization assay or blocks the inhibitory effect ofsclerostin in Smad1 phosphorylation assay or the antibody inhibitsbinding of sclerostin to the LRP-6 or the antibody increases boneformation and mass and density.

In various embodiments, the antibody may exhibit one or more, two ormore, or three or more of the functional properties listed discussedabove. Such antibodies can be, for example, human antibodies, humanizedantibodies or chimeric antibodies.

In other embodiments, an antibody of the invention optimized forexpression in a mammalian cell has a full length heavy chain sequenceand a full length light chain sequence, wherein one or more of thesesequences have specified amino acid sequences based on the antibodiesdescribed herein or conservative modifications thereof, and wherein theantibodies retain the desired functional properties of theanti-sclerostin antibodies of the invention. Accordingly, the inventionprovides an isolated monoclonal antibody optimized for expression in amammalian cell consisting of a full length heavy chain and a full lengthlight chain wherein: the full length heavy chain has amino acidsequences selected from the group of SEQ ID NOs: 111-121, andconservative modifications thereof; and the full length light chain hasamino acid sequences selected from the group of SEQ ID NOs: 122-132, andconservative modifications thereof; the antibody specifically binds tosclerostin; and the antibody exhibits at least one of the followingfunctional properties: the antibody blocks the inhibitory effect ofsclerostin in a cell based wnt signaling assay, the antibody blocks theinhibitory effect of sclerostin in a cell based mineralization assay orblocks the inhibitory effect of sclerostin in Smad1 phosphorylationassay or the antibody inhibits binding of sclerostin to the LRP-6 or theantibody increases bone formation and mass and density.

In various embodiments, the antibody may exhibit one or more, two ormore, or three or more of the functional properties listed discussedabove. Such antibodies can be, for example, human antibodies, humanizedantibodies or chimeric antibodies.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one ormore amino acid residues within the CDR regions of an antibody of theinvention can be replaced with other amino acid residues from the sameside chain family, and the altered antibody can be tested for retainedfunction using the functional assays described herein.

Antibodies that Bind to the Same Epitope as Anti-Sclerostin Antibodiesof the Invention

In another embodiment, the invention provides antibodies that bind tothe same epitope as do the various specific anti-sclerostin antibodiesof the invention described herein. It has indeed been surprisingly foundthat all the antibodies described in the Examples that are capable of:

(i) blocking the inhibitory effect of sclerostin in a cell based wntsignaling assay;

(ii) blocking the inhibitory effect in a cell based mineralizationassay;

(iii) inhibiting binding of sclerostin to the LRP-6; and,

(iv) increasing bone formation, mass and density,

bind the same epitope in sclerostin with high affinity, said epitopebeing a conformational epitope that include amino acids from both SEQ IDNO:156 and SEQ ID NO:157. Without being bound by any specific model, itis proposed here that the amino acid sequences SEQ ID NO:156 and SEQ IDNO:157 delineate one conformational epitope region in the sclerostinpolypeptide that is recognized by the antibodies of the invention.

Additional antibodies can therefore be identified based on their abilityto cross-compete (e.g., to competitively inhibit the binding of, in astatistically significant manner) with other antibodies of the inventionin standard sclerostin binding assays. The ability of a test antibody toinhibit the binding of antibodies of the present invention to humansclerostin demonstrates that the test antibody can compete with thatantibody for binding to human sclerostin; such an antibody may,according to non-limiting theory, bind to the same or a related (e.g., astructurally similar or spatially proximal) epitope on human sclerostinas the antibody with which it competes. In a certain embodiment, theantibody that binds to the same epitope on human sclerostin as theantibodies of the present invention is a human monoclonal antibody. Suchhuman monoclonal antibodies can be prepared and isolated as described inthe Examples.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the VH and/or VL sequences shown herein asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., VH and/or VL), for example within one ormore CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedmonoclonal antibody, or a functional protein comprising an antigenbinding portion thereof, comprising a heavy chain variable regioncomprising CDR1 sequences having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1-11; CDR2 sequences having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 12-22;CDR3 sequences having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 23-33, respectively; and a light, chainvariable region having CDR1 sequences having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 34-44; CDR2 sequenceshaving an amino acid sequence selected from the group consisting of SEQID NOs: 45-55; and CDR3 sequences consisting of an amino acid sequenceselected from the group consisting of SEQ ID NOs: 56-66, respectively.Thus, such antibodies contain the V_(H) and V_(L) CDR sequences ofmonoclonal antibodies, yet may contain different framework sequencesfrom these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al., 1991 Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.,1992 J. fol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. JImmunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

An example of framework sequences for use in the antibodies of theinvention are those that are structurally similar to the frameworksequences used by selected antibodies of the invention, e.g., consensussequences and/or framework sequences used by monoclonal antibodies ofthe invention. The V_(H) CDR1, 2 and 3 sequences, and the V_(L) CDR1, 2and 3 sequences, can be grafted onto framework regions that have theidentical sequence as that found in the germline immunoglobulin genefrom which the framework sequence derive, or the CDR sequences can begrafted onto framework regions that contain one or more mutations ascompared to the germline sequences. For example, it has been found thatin certain instances it is beneficial to mutate residues within theframework regions to maintain or enhance the antigen binding ability ofthe antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Accordingly, in another embodiment, the invention provides isolatedanti-sclerostin monoclonal antibodies, or a functional proteincomprising an antigen binding portion thereof, consisting of a heavychain variable region having: a V_(H) CDR1 region consisting of an aminoacid sequence selected from the group having SEQ ID NOs: 1-11 or anamino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 1-11; aV_(H) CDR2 region having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 12-22, or an amino acid sequence having one,two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 12-22; a V_(H) CDR3 region havingan amino acid sequence selected from the group consisting of SEQ ID NOs:23-33, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 23-33; a V_(L) CDR1 region having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 34-44, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 34-44; a V_(L) CDR2region having an amino acid sequence selected from the group consistingof SEQ ID NOs: 45-55, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 45-55; and a V_(L) CDR3 region having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 56-66,or an amino acid sequence having one, two, three, four or five aminoacid substitutions, deletions or additions as compared to SEQ ID NOs:56-66.

Grafting Antigen-Binding Domains Into Alternative Frameworks orScaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region which specifically binds to sclerostin. Such frameworksor scaffolds include the 5 main idiotypes of human immunoglobulins, orfragments thereof (such as those disclosed elsewhere herein), andinclude immunoglobulins of other animal species, preferably havinghumanized aspects. Single heavy-chain antibodies such as thoseidentified in camelids are of particular interest in this regard. Novelframeworks, scaffolds and fragments continue to be discovered anddeveloped by those skilled in the art.

In one aspect, the invention pertains to generating non-immunoglobulinbased antibodies using non-immunoglobulin scaffolds onto which CDRs ofthe invention can be grafted. Known or future non-immunoglobulinframeworks and scaffolds may be employed, as long as they comprise abinding region specific for the target protein of SEQ ID NO: 155. Suchcompounds are known herein as “polypeptides comprising a target-specificbinding region”. Examples of non-immunoglobulin framework are furtherdescribed in the sections below (camelid antibodies and non-antibodyscaffold).

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Camelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variabledomain identified as V_(HH) can be obtained by genetic engineering toyield a small protein having high affinity for a target, resulting in alow molecular weight antibody-derived protein known as a “camelidnanobody”. See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see alsoStijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. etal., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 BioconjugateChem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89:456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineeredlibraries of camelid antibodies and antibody fragments are commerciallyavailable, for example, from Ablynx, Ghent, Belgium. As with otherantibodies of non-human origin, an amino acid sequence of a camelidantibody can be altered recombinantly to obtain a sequence that moreclosely resembles a human sequence, i.e., the nanobody can be“humanized”. Thus the natural low antigenicity of camelid antibodies tohumans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule, and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. patent application20040161738 published Aug. 19, 2004. These features combined with thelow antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins with bacteriophageand are functional.

Accordingly, a feature of the present invention is a camelid antibody ornanobody having high affinity for sclerostin. In certain embodimentsherein, the camelid antibody or nanobody is naturally produced in thecamelid animal, i.e., is produced by the camelid following immunizationwith sclerostin or a peptide fragment thereof, using techniquesdescribed herein for other antibodies. Alternatively, theanti-sclerostin camelid nanobody is engineered, i.e., produced byselection for example from a library of phage displaying appropriatelymutagenized camelid nanobody proteins using panning procedures withsclerostin as a target as described in the examples herein. Engineerednanobodies can further be customized by genetic engineering to have ahalf life in a recipient subject of from 45 minutes to two weeks. In aspecific embodiment, the camelid antibody or nanobody is obtained bygrafting the CDRs sequences of the heavy or light chain of the humanantibodies of the invention into nanobody or single domain antibodyframework sequences, as described for example in PCT/EP93/02214(WO94/04678).

Non-Antibody Scaffold

Known non-immunoglobulin frameworks or scaffolds include, but are notlimited to, Adnectins (fibronectin) (Compound Therapeutics, Inc.,Waltham, Mass.), ankyrin (Molecular Partners AG, Zurich, Switzerland),domain antibodies (Domantis, Ltd (Cambridge, Mass.) and Ablynx nv(Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG,Freising, Germany), small modular immuno-pharmaceuticals (TrubionPharmaceuticals Inc., Seattle, Wash.), maxybodies (Avidia, Inc.(Mountain View, Calif.)), Protein A (Affibody AG, Sweden) and affilin(gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany),protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

(i) Adnectins—Compound Therapeutics

The adnectin scaffolds are based on fibronectin type III domain (e.g.,the tenth module of the fibronectin type III (10 Fn3 domain)). Thefibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (U.S. Pat. No.6,818,418).

These fibronectin-based scaffolds are not an immunoglobulin, althoughthe overall fold is closely related to that of the smallest functionalantibody fragment, the variable region of the heavy chain, whichcomprises the entire antigen recognition unit in camel and llama IgG.Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of the invention using standardcloning techniques.

(ii) Ankyrin—Molecular Partners

The technology is based on using proteins with ankyrin derived repeatmodules as scaffolds for bearing variable regions which can be used forbinding to different targets. The ankyrin repeat module is a 33 aminoacid polypeptide consisting of two anti-parallel α-helices and a β-turn.Binding of the variable regions is mostly optimized by using ribosomedisplay.

(iii) Maxybodies/Avimers—Avidia

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example,20040175756; 20050053973; 20050048512; and 20060008844.

(vi) Protein A—Affibody

Affibody® affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate Affibody® libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibody®molecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of Affibody® molecules issimilar to that of an antibody.

(v) Anticalins—Pieris

Anticalins® are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids.

The protein architecture is reminiscent of immunoglobulins, withhypervariable loops on top of a rigid framework. However, in contrastwith antibodies or their recombinant fragments, lipocalins are composedof a single polypeptide chain with 160 to 180 amino acid residues, beingjust marginally bigger than a single immunoglobulin domain.

The set of four loops, which makes up the binding pocket, showspronounced structural plasticity and tolerates a variety of side chains.The binding site can thus be reshaped in a proprietary process in orderto recognize prescribed target molecules of different shape with highaffinity and specificity.

One protein of lipocalin family, the bilin-binding protein (BBP) ofPieris brassicae has been used to develop anticalins by mutagenizing theset of four loops. One example of a patent application describing“anticalins” is PCT WO199916873.

(vi) Affilin—Scil Proteins

Affilin™ molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New Affilin™ molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.

Affilin™ molecules do not show any structural homology to immunoglobulinproteins. Scil Proteins employs two Affilin™ scaffolds, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368

(vii) Protein Epitope Mimetics (PEM)

PEM are medium-sized, cyclic, peptide-like molecules (MW 1-2 kDa)mimicking beta-hairpin secondary structures of proteins, the majorsecondary structure involved in protein-protein interactions. Moregenerally, any polypeptide that mimicks the 3D structure of the epitopeof the disclosed antibodies are part of the present invention. Preferredembodiments are polypeptides of 30-100 amino acids comprising at leastthe following polypeptides E1-L-E2, wherein E1 is SEQ ID NO:156 and E2is SEQ ID NO:157 and L is an polypeptidic linker allowing E1 and E2 toreproduce the 3D structure of the region recognized by the antibodies ofthe invention. According to a preferred embodiment, L is a linkerconsisting of 10-20 amino acids selected among glycine or serine aminoacids. Preferably the linker L comprises the peptide GGGSGGGGSGGGG (SEQID NO: X/SEQ ID NO: 158) or GGGGSGGGGSGGGGSGGGG (SEQ ID NO:Y/SEQ ID NO:159), more preferably the linker L is consisting essentially of SEQ IDNO: X or SEQ ID NO:Y.

These polypeptides should retain high affinity to the antibodies of theinvention. These polypeptides can also be advantageously used asimmunogens to raise antibodies against sclerostin.

These polypeptides can also be used as antagonist or agonist ofsclerostin and therefore have similar applications as those describedfor the antibodies of the present invention.

Polypeptides with one or more amino acid substitutions or deletions,preferably not less than 1, 2 or 3 amino acid substitutions, ordeletions in E1 and/or E2 sequence, are also part of the invention.These polypeptides may further be engineered to increase half life orimprove solubility. Especially, fusion constructs of these polypeptideswith serum proteins, such as Fc fragments of IgG or human serum albumincan be generated to increase half life, similarly to Fc engineeringdescribed in the following paragraph for antibody fragment molecules ofthe invention.

Framework or Fc Engineering

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis. Such “backmutated” antibodiesare also intended to be encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell-epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret al.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for “antigen”. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lecl3 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Another modification of the antibodies herein that is contemplated bythe invention is pegylation. An antibody can be pegylated, for example,to increase the biological (e.g., serum) half-life of the antibody. Topegylate an antibody, the antibody, or fragment thereof, typically isreacted with polyethylene glycol (PEG), such as a reactive ester oraldehyde derivative of PEG, under conditions in which one or more PEGgroups become attached to the antibody or antibody fragment. Thepegylation can be carried out by an acylation reaction or an alkylationreaction with a reactive PEG molecule (or an analogous reactivewater-soluble polymer). As used herein, the term “polyethylene glycol”is intended to encompass any of the forms of PEG that have been used toderivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the invention. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Another modification of the antibodies that is contemplated by theinvention is a conjugate or a protein fusion of at least theantigen-binding region of the antibody of the invention to serumprotein, such as human serum albumin or a fragment thereof to increasehalf-life of the resulting molecule. Such approach is for exampledescribed in Ballance et al. EP0322094.

Another possibility is a fusion of at least the antigen-binding regionof the antibody of the invention to proteins capable of binding to serumproteins, such human serum albumin to increase half life of theresulting molecule. Such approach is for example described in Nygren atal., EP 0 486 525.

Methods of Engineering Altered Antibodies

As discussed above, the anti-sclerostin antibodies having V_(H) andV_(L) sequences or full length heavy and light chain sequences shownherein can be used to create new anti-sclerostin antibodies by modifyingfull length heavy chain and/or light chain sequences, V_(H) and/or V_(L)sequences, or the constant region(s) attached thereto. Thus, in anotheraspect of the invention, the structural features of an anti-sclerostinantibody of the invention are used to create structurally relatedanti-sclerostin antibodies that retain at least one functional propertyof the antibodies of the invention, such as binding to human sclerostinand also inhibiting one or more functional properties of sclerostin(e.g., receptor binding, preventing or ameliorating osteolysis).

For example, one or more CDR regions of the antibodies of the presentinvention, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, anti-sclerostin antibodies of the invention,as discussed above. Other types of modifications include those describedin the previous section. The starting material for the engineeringmethod is one or more of the V_(H) and/or V_(L) sequences providedherein, or one or more CDR regions thereof. To create the engineeredantibody, it is not necessary to actually prepare (i.e., express as aprotein) an antibody having one or more of the V_(H) and/or V_(L)sequences provided herein, or one or more CDR regions thereof. Rather,the information contained in the sequence(s) is used as the startingmaterial to create a “second generation” sequence(s) derived from theoriginal sequence(s) and then the “second generation” sequence(s) isprepared and expressed as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-sclerostin antibody consisting of: a heavy chainvariable region antibody sequence having a CDR1 sequence selected fromthe group consisting of SEQ ID NOs: 1-11, a CDR2 sequence selected fromthe group consisting of SEQ ID NOs: 12-22 and/or a CDR3 sequenceselected from the group consisting of SEQ ID NOs: 23-33; and a lightchain variable region antibody sequence having a CDR1 sequence selectedfrom the group consisting of SEQ ID NOs: 34-44, a CDR2 sequence selectedfrom the group consisting of SEQ ID NOs: 45-55 and/or a CDR3 sequenceselected from the group consisting of SEQ ID NOs: 56-66; altering atleast one amino acid residue within the heavy chain variable regionantibody sequence and/or the light chain variable region antibodysequence to create at least one altered antibody sequence; andexpressing the altered antibody sequence as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-sclerostin antibody optimized for expression in amammalian cell consisting of: a full length heavy chain antibodysequence having a sequence selected from the group of SEQ ID NOs:111-121; and a full length light chain antibody sequence having asequence selected from the group of 122-132; altering at least one aminoacid residue within the full length heavy chain antibody sequence and/orthe full length light chain antibody sequence to create at least onealtered antibody sequence; and expressing the altered antibody sequenceas a protein.

The altered antibody sequence can also be prepared by screening antibodylibraries having fixed CDR3 sequences selected among the groupconsisting of SEQ ID NO:23-33 or minimal essential binding determinantsas described in US20050255552 and diversity on CDR1 and CDR2 sequences.The screening can be performed according to any screening technologyappropriate for screening antibodies from antibody libraries, such asphage display technology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the anti-sclerostin antibodies describedherein, which functional properties include, but are not limited to,specifically binding to human sclerostin; and the antibody exhibits atleast one of the following functional properties: the antibody blocksthe inhibitory effect of sclerostin in a cell based wnt signaling assay,the antibody blocks the inhibitory effect of sclerostin in a cell basedmineralization assay or blocks the inhibitory effect of sclerostin inSmad1 phosphorylation assay or the antibody inhibits binding ofsclerostin to the LRP-6 or the antibody increases bone formation andmass and density.

The altered antibody may exhibit one or more, two or more, or three ormore of the functional properties discussed above.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., ELISAs).

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an anti-sclerostin antibody coding sequence and the resultingmodified anti-sclerostin antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention pertains to nucleic acid molecules thatencode the antibodies of the invention. Examples of full length lightchain parental nucleotide sequences are shown in SEQ ID NOs 144-145.Examples of full length heavy chain parental nucleotide sequences areshown in SEQ ID NOs 133-134. Examples of full length light chainnucleotide sequences optimized for expression in a mammalian cell areshown in SEQ ID NOs: 146-154. Examples of full length heavy chainnucleotide sequences optimized for expression in a mammalian cell areshown in SEQ ID NOs: 135-143.

The nucleic acids may be present in whole cells, in a cell lysate, ormay be nucleic acids in a partially purified or substantially pure form.A nucleic acid is “isolated” or “rendered substantially pure” whenpurified away from other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis and others well known in the art. See, F.Ausubel, et al., ed. 1987 Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid of theinvention can be, for example, DNA or RNA and may or may not containintronic sequences. In an embodiment, the nucleic acid is a cDNAmolecule. The nucleic acid may be present in a vector such as a phagedisplay vector, or in a recombinant plasmid vector.

Nucleic acids of the invention can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromvarious phage clones that are members of the library.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to an scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA molecule, or to a fragment encodinganother protein, such as an antibody constant region or a flexiblelinker. The term “operatively linked”, as used in this context, isintended to mean that the two DNA fragments are joined in a functionalmanner, for example, such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame, or such that the protein is expressedunder control of a desired promoter.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., et al., 1991 Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavychain constant region is selected among IgG2 isotypes. For a Fabfragment heavy chain gene, the V_(H)-encoding DNA can be operativelylinked to another DNA molecule encoding only the heavy chain CH1constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as to a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al., 1991 Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or a lambda constant region.

To create an scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly4-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(H) and V_(H) regions joined by the flexible linker (seee.g., Bird et al., 1988 Science 242:423-426; Huston et al., 1988 Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature348:552-554).

Generation of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,1975 Nature 256: 495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

An animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a well established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art. See e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.

In a certain embodiment, the antibodies of the invention are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstsclerostin can be generated using transgenic or transchromosomic micecarrying parts of the human immune system rather than the mouse system.These transgenic and transchromosomic mice include mice referred toherein as HuMAb mice and KM mice, respectively, and are collectivelyreferred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.,1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N.,1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D., 1995 Intern. Rev. Immunol 13: 65-93, and Harding, F. andLonberg, N., 1995 Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMAb mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al., 1992 Nucleic AcidsResearch 20:6287-6295; Chen, J. et al., 1993 International Immunology 5:647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94:3720-3724;Choi et al., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBOJ. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor,L. et al., 1994 International Immunology 579-591; and Fishwild, D. etal., 1996 Nature Biotechnology 14: 845-851, the contents of all of whichare hereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92/103918, WO 93/12227, WO 94/25585, WO 97/113852,WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchromosomes such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-sclerostin antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused. Such mice are described in, e.g., U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-sclerostin antibodies of the invention. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranschromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., 2002Nature Biotechnology 20:889-894) and can be used to raiseanti-sclerostin antibodies of the invention.

Human monoclonal antibodies of the invention can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art or described in the examples below. See forexample: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner etal.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat.Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos.5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 toGriffiths et al.

Human monoclonal antibodies of the invention can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Generation of Hybridomas Producing Human Monoclonal Antibodies

To generate hybridomas producing human monoclonal antibodies of theinvention, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells(ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×145in flat bottom microtiter plates, followed by a two week incubation inselective medium containing 20% fetal Clone Serum, 18% “653” conditionedmedia, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mMHEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/mlstreptomycin, 50 mg/ml gentamycin and 1× HAT (Sigma; the HAT is added 24hours after the fusion). After approximately two weeks, cells can becultured in medium in which the HAT is replaced with HT. Individualwells can then be screened by ELISA for human monoclonal IgM and IgGantibodies. Once extensive hybridoma growth occurs, medium can beobserved usually after 10-14 days. The antibody secreting hybridomas canbe replated, screened again, and if still positive for human IgG, themonoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the CH segment(s) within the vector andthe V_(L) segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Regulatory sequences for mammalian host cell expression includeviral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., theadenovirus major late promoter (AdMLP)), and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or P-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRa promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al., 1988 Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. It is theoretically possible toexpress the antibodies of the invention in either prokaryotic oreukaryotic host cells. Expression of antibodies in eukaryotic cells, inparticular mammalian host cells, is discussed because such eukaryoticcells, and in particular mammalian cells, are more likely thanprokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss, M. A. and Wood, C. R., 1985 Immunology Today6:12-13).

Mammalian host cells for expressing the recombinant antibodies of theinvention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHOcells, described Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA77:4216-4220 used with a DH FR selectable marker, e.g., as described inR. J. Kaufman and P. A. Sharp, 1982 Mol. Biol. 159:601-621, NSO myelomacells, COS cells and SP2 cells. In particular, for use with NSO myelomacells, another expression system is the GS gene expression system shownin WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or secretion of the antibody into the culture medium in whichthe host cells are grown. Antibodies can be recovered from the culturemedium using standard protein purification methods.

Bispecific Molecules

In another aspect, the present invention features bispecific ormultispecific molecules comprising an anti-sclerostin antibody, or afragment thereof, of the invention. An antibody of the invention, orantigen-binding regions thereof, can be derivatized or linked to anotherfunctional molecule, e.g., another peptide or protein (e.g., anotherantibody or ligand for a receptor) to generate a bispecific moleculethat binds to at least two different binding sites or target molecules.The antibody of the invention may in fact be derivatized or linked tomore than one other functional molecule to generate multi-specificmolecules that bind to more than two different binding sites and/ortarget molecules; such multi-specific molecules are also intended to beencompassed by the term “bispecific molecule” as used herein. To createa bispecific molecule of the invention, an antibody of the invention canbe functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for sclerostin and asecond binding specificity for a second target epitope. For example, thesecond target epitope is another epitope of sclerostin different fromthe first target epitope. Another example is a bispecific moleculecomprising at least one first binding specificity for sclerostin and asecond binding specificity for an epitope within Dkk-1. Another exampleis a bispecific molecule comprising at least one first bindingspecificity for sclerostin and a second binding specificity for anepitope within LRP4.

Additionally, for the invention in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to the first and second target epitope.

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

Other antibodies which can be employed in the bispecific molecules ofthe invention are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities, using methods knownin the art. For example, each binding specificity of the bispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-I-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83, and Glennie et al.,1987 J. Immunol. 139: 2367-2375. Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

Multivalent Antibodies

In another aspect, the present invention provides multivalent compoundscomprising at least two identical or different antigen-binding portionsof the antibodies of the invention binding to sclerostin. Preferably,given the trimeric nature of sclerostin, the compounds of the inventionprovides at least three or four antigen-binding portions of theantibodies. The antigen-binding portions can be linked together viaprotein fusion or covalent or non covalent linkage. Alternatively,methods of linkage has been described for the bispecfic molecules.Tetravalent compounds can be obtained for example by cross-linkingantibodies of the antibodies of the invention with an antibody thatbinds to the constant regions of the antibodies of the invention, forexample the Fc or hinge region.

Trimerizing domain are described for example in Borean patent EP 1 012280B1. Pentamerizing modules are described for example inPCT/EP97/05897.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofmonoclonal antibodies, or antigen-binding region(s) thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies, or immunoconjugates orbispecific molecules of the invention. For example, a pharmaceuticalcomposition of the invention can comprise a combination of antibodiesthat bind to different epitopes on the target antigen or that havecomplementary activities.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-sclerostin antibody of thepresent invention combined with at least one other anti-inflammatory oranti-osteoporotic agent. Examples of therapeutic agents that can be usedin combination therapy are described in greater detail below in thesection on uses of the antibodies of the invention. The compositions arepreferably formulated at physiological pH.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier should be suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjuage, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al., 1977 J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, andthe like; and metal chelating agents, such as citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, one can include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent that delays absorption for example, monostearatesalts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation are vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, from about 0.1percent to about 70 percent, or from about 1 percent to about 30 percentof active ingredient in combination with a pharmaceutically acceptablecarrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Dosage regimens for an anti-sclerostin antibodyof the invention include 1 mg/kg body weight or 3 mg/kg body weight byintravenous administration, with the antibody being given using one ofthe following dosing schedules: every four weeks for six dosages, thenevery three months; every three weeks; 3 mg/kg body weight once followedby 1 mg/kg body weight every three weeks.

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously or sequentially,in which case the dosage of each antibody administered falls within theranges indicated. Antibody is usually administered on multipleoccasions. Intervals between single dosages can be, for example, weekly,monthly, every three months or yearly. Intervals can also be irregularas indicated by measuring blood levels of antibody to the target antigenin the patient. In some methods, dosage is adjusted to achieve a plasmaantibody concentration of about 1-1000 μg/ml and in some methods about25-300 μg/ml.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated or until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-sclerostin antibody ofthe invention can result in a decrease in severity of disease symptoms,an increase in frequency and duration of disease symptom-free periods,or a prevention of impairment or disability due to the diseaseaffliction.

A composition of the present invention can be administered by one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Routes of administration for antibodies of the inventioninclude intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Alternatively, an antibody of the invention can be administered by anonparenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in one embodiment, a therapeutic composition ofthe invention can be administered with a needleless hypodermic injectiondevice, such as the devices shown in U.S. Pat. Nos. 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.Examples of well known implants and modules useful in the presentinvention include: U.S. Pat. No. 4,487,603, which shows an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which shows a therapeutic device for administeringmedicants through the skin; U.S. Pat. No. 4,447,233, which shows amedication infusion pump for delivering medication at a precise infusionrate; U.S. Pat. No. 4,447,224, which shows a variable flow implantableinfusion apparatus for continuous drug delivery; U.S. Pat. No.4,439,196, which shows an osmotic drug delivery system havingmulti-chamber compartments; and U.S. Pat. No. 4,475,196, which shows anosmotic drug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade,1989 J. Cline Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., 1988 Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al., 1995 FEBS Lett. 357:140; M.Owais et al., 1995 Antimicrob. Agents Chernother. 39:180); surfactantprotein A receptor (Briscoe et al., 1995 Am. J. Physiol. 1233:134); p120(Schreier et al., 1994 J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen, 1994 FEBS Lett. 346:123; J. J. Killion; I. J. Fidler,1994 Immnunomethods 4:273.

Uses and Methods of the Invention

The antibodies of the present invention have in vitro and in vivodiagnostic and therapeutic utilities. For example, these molecules canbe administered to cells in culture, e.g. in vitro or in vivo, or in asubject, e.g., in vivo, to treat, prevent or diagnose a variety ofdisorders. The term “subject” as used herein is intended to includehuman and non-human animals. Non-human animals includes all vertebrates,e.g., mammals and non-mammals, such as non-human primates, sheep, dogs,cats, cows, horses, chickens, amphibians, and reptiles.

The methods are particularly suitable for treating, preventing ordiagnosing sclerostin-related disorders and/or aberrant bone mineraldensity disorders, e.g., osteoporosis.

The invention also provides methods for increasing the mineral contentand/or mineral density of bone. Compositions of the present inventionmay also be useful for improving outcomes in orthopedic procedures,dental procedures, implant surgery, joint replacement, bone grafting,bone cosmetic surgery and bone repair such as fracture healing, nonunionhealing, delayed union healing and facial reconstruction. One or morecompositions may be administered before, during and/or after theprocedure, replacement, graft, surgery or repair.

As used herein, “a sclerostin-related disorder” includes disorders inwhich bone mineral density (BMD) is abnormally and/or pathologically lowrelative to healthy subjects. Disorders characterized by low BMD and/orbone fragility include but are not limited to primary and secondaryosteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta (OI),avascular necrosis (osteonecrosis), fractures and implant healing(dental implants and hip implants), bone loss due to other disorders(e.g., associated with HIV infection, cancers, or arthritis). Other“sclerostin-related disorders” include but are not limited to rheumatoidarthritis, osteoarthritis, arthritis, and the formation and/or presenceof osteolytic lesions.

As used herein, “a sclerostin-related disorder” includes conditionsassociated with or characterized by aberrant sclerostin levels. Theseinclude cancers and osteoporotic conditions (e.g., osteoporosis orosteopenia), some of which overlap with “sclerostin-related disorders”as defined herein. Sclerostin-related cancers can include myeloma (e.g.,multiple myeloma with osteolytic lesions), breast cancer, colon cancer,melanoma, hepatocellular cancer, epithelial cancer, esophageal cancer,brain cancer, lung cancer, prostate cancer, or pancreatic cancer, aswell as any metastases thereof.

A “sclerostin-related disorder” can also include renal andcardiovascular conditions, due at least to sclerostin's expression inthe kidney and cardiovasculature. Said disorders include but are notlimited to such renal disorders as glomerular diseases (e.g., acute andchronic glomerulonephritis, rapidly progressive glomerulonephritis,nephrotic syndrome, focal proliferative glomerulonephritis, glomerularlesions associated with systemic disease, such as systemic lupuserythematosus, Goodpasture's syndrome, multiple myeloma, diabetes,polycystic kidney disease, neoplasia, sickle cell disease, and chronicinflammatory diseases), tubular diseases (e.g., acute tubular necrosisand acute renal failure, polycystic renal diseasemedullary spongekidney, medullary cystic disease, nephrogenic diabetes, and renaltubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis,drug and toxin induced tubulointerstitial nephritis, hypercalcemicnephropathy, and hypokalemic nephropathy) acute and rapidly progressiverenal failure, chronic renal failure, nephrolithiasis, gout, vasculardiseases (e.g., hypertension and nephrosclerosis, microangiopathichemolytic anemia, atheroembolic renal disease, diffuse corticalnecrosis, and renal infarcts), or tumors (e.g., renal cell carcinoma andnephroblastoma).

Said disorders also include but are not limited to such cardiovasculardisorders as ischemic heart disease (e.g., angina pectoris, myocardialinfarction, and chronic ischemic heart disease), hypertensive heartdisease, pulmonary heart disease, valvular heart disease (e.g.,rheumatic fever and rheumatic heart disease, endocarditis, mitral valveprolapse, and aortic valve stenosis), congenital heart disease (e.g.,valvular and vascular obstructive lesions, atrial or ventricular septaldefect, and patent ductus arteriosus), or myocardial disease (e.g.,myocarditis, congestive cardiomyopathy, and hypertrophic cariomyopathy).

When antibodies to sclerostin are administered together with anotheragent, the two can be administered in either order (i.e. sequentially)or simultaneously.

According to a further embodiment of the invention, the antibodies ofthe invention may be employed as adjunct or adjuvant to other therapy,e.g. a therapy using a bone resorption inhibitor, for example as inosteoporosis therapy, in particular a therapy employing calcium, acalcitonin or an analogue or derivative thereof, e.g. salmon, eel orhuman calcitonin, calcilytics, calcimimetics (e.g., cinacalcet), asteroid hormone, e.g. an estrogen, a partial estrogen agonist orestrogen-gestagen combination, a SERM (Selective Estrogen ReceptorModulator) e.g. raloxifene, lasofoxifene, bazedoxifene, arzoxifene,FC1271, Tibolone (Livial®), a SARM (Selective Androgen ReceptorModulator), a RANKL antibody (such as denosumab), a cathepsin Kinhibitor, vitamin D or an analogue thereof or PTH, a PTH fragment or aPTH derivative e.g. PTH (1-84) (such as Preos™), PTH (1-34) (such asForteo™), PTH (1-36), PTH (1-38), PTH (1-31)NH2 or PTS 893. According toanother embodiment, the antibodies of the invention may be employed incombination with other current osteoporosis therapy approaches,including bisphosphonates (e.g., Fosamax™ (alendronate), Actonel™(risedronate sodium), Bonviva™ (ibandronic acid), Zometa™ (zoledronicacid), Aclasta™/Reclast™ (zoledronic acid), olpadronate, neridronate,skelid, bonefos), statins, anabolic steroids, lanthanum and strontiumsalts, and sodium fluoride.

In one specific embodiment, the antibodies of the invention may beadministered in combination with an LRP4 modulating agent, i.e., anagent modulating the expression or activity of LRP4, e.g, an LRP4neutralizing antibody.

In another specific embodiment, the antibodies of the invention may beadministered in combination with a DKK1 modulating agent, i.e., an agentthat interfere or neutralize Dkk-1 mediated antagonism of Wnt signaling,e.g., a DKK1 neutralizing antibody.

Thus, the invention also provides the use of an antibody or functionalprotein of the invention and (i) zoledronic acid, (ii) an anti-DKK1antibody, (iii) alendronate, (iv) an anti-LRP4 antibody, (v) hPTH and/or(vi) parathyroid hormone releasing agents (calcilytics), in themanufacture of a medicament for the treatment of a pathological disorderthat is mediated by sclerostin or that is associated with an increasedlevel of sclerostin.

In another embodiment, the invention provides the use of an antibody orfunctional protein of the invention in the manufacture of a medicamentfor the treatment of a pathological disorder that is mediated bysclerostin or that is associated with an increased level of sclerostin,wherein the medicament is used in conjunction with (i) zoledronic acid,(ii) an anti-DKK1 antibody, (iii) alendronate, (iv) an anti-LRP4antibody, (v) hPTH and/or (vi) parathyroid hormone releasing agents(calcilytics).

In another embodiment, the invention provides the use of (i) zoledronicacid, (ii) an anti-DKK1 antibody, (iii) alendronate, (iv) an anti-LRP4antibody, (v) hPTH and/or (vi) parathyroid hormone releasing agents(calcilytics), in the manufacture of a medicament for the treatment of apathological disorder that is mediated by sclerostin or that isassociated with an increased level of sclerostin, wherein the medicamentis used in conjunction with an antibody or functional protein of theinvention.

In another embodiment, the invention provides the use of an antibody orfunctional protein of the invention in the manufacture of a medicamentfor the treatment of a pathological disorder that is mediated bysclerostin or that is associated with an increased level of sclerostin,wherein the patient has been pre-administered with (i) zoledronic acid,(ii) an anti-DKK1 antibody, (iii) alendronate, (iv) an anti-LRP4antibody, (v) hPTH and/or (vi) parathyroid hormone releasing agents(calcilytics).

In another embodiment, the invention provides the use of (i) zoledronicacid, (ii) an anti-DKK1 antibody, (iii) alendronate, (iv) an anti-LRP4antibody, (v) hPTH and/or (vi) parathyroid hormone releasing agents(calcilytics), in the manufacture of a medicament for the treatment of apathological disorder that is mediated by sclerostin or that isassociated with an increased level of sclerostin, wherein the patienthas been pre-administered with an antibody or functional protein of theinvention.

In one embodiment of the combinations recited above, the hPTH ishPTH(1-34).

In one embodiment, the antibodies of the invention can be used to detectlevels of sclerostin, or levels of cells that contain sclerostin. Thiscan be achieved, for example, by contacting a sample (such as an invitro sample) and a control sample with the anti-sclerostin antibodyunder conditions that allow for the formation of a complex between theantibody and sclerostin. Any complexes formed between the antibody andsclerostin are detected and compared in the sample and the control. Forexample, standard detection methods, well known in the art, such asELISA and flow cytometic assays, can be performed using the compositionsof the invention.

Accordingly, in one aspect, the invention further provides methods fordetecting the presence of sclerostin (e.g., human sclerostin antigen) ina sample, or measuring the amount of sclerostin, comprising contactingthe sample, and a control sample, with an antibody of the invention, oran antigen binding region thereof, which specifically binds tosclerostin, under conditions that allow for formation of a complexbetween the antibody or portion thereof and sclerostin. The formation ofa complex is then detected, wherein a difference in complex formationbetween the sample compared to the control sample is indicative of thepresence of sclerostin in the sample.

Also within the scope of the invention are kits consisting of thecompositions (e.g., antibodies, human antibodies and bispecificmolecules) of the invention and instructions for use. The kit canfurther contain a least one additional reagent, or one or moreadditional antibodies of the invention (e.g., an antibody having acomplementary activity which binds to an epitope on the target antigendistinct from the first antibody). For example, such kits may comprisean antibody or functional protein of the invention and one or more of(i) zoledronic acid, (ii) an anti-DKK1 antibody, (iii) alendronate, (iv)an anti-LRP4 antibody, (v) hPTH and (vi) parathyroid hormone releasingagents (calcilytics). Kits typically include a label indicating theintended use of the contents of the kit. The term label includes anywriting, or recorded material supplied on or with the kit, or whichotherwise accompanies the kit.

The invention having been fully described, it is further illustrated bythe following examples and claims, which are illustrative and are notmeant to be further limiting. Those skilled in the art will recognize orbe able to ascertain using no more than routine experimentation,numerous equivalents to the specific procedures described herein. Suchequivalents are within the scope of the present invention and claims.The contents of all references, including issued patents and publishedpatent applications, cited throughout this application are herebyincorporated by reference.

EXAMPLES Functional Assays

Alkaline Phosphatase Assay (ALP)

Cells

MC3T3 1b cells is a clone of MC3T3-JP cells expressing OSE2-luc. Thisclone was obtained after stable transfection of MC3T3-JP (MC3T3-E1 mouseosteoblast, Japan clone, Jp; a kind gift of Dr. T. Kokkubo, Novartis,Japan) using 8x-OSE2wt-mOG2luc in pcDNA3.1+.

Culture Medium

MC3T3-1 b cells were routinely cultivated in minimum essential mediumalpha (MEMα; Invitrogen, Cat #22561-021) supplemented with 10% fetalcalf serum (FCS; Amimed Cat #2-01F100-I), 2 mM L-glutamine (Gibco Cat#25030-024), 50 IU penicillin/50 μg/ml streptomycin (Amimed Cat#4-01F00-H) and 10 mM Hepes (Gibco Cat #15630-056) and 0.75 mg/ml G418(Gibco Cat #10131-027) (maintenance culture medium).

Stock Solutions

Ten mM ascorbic acid (Wako Pure Chemical Cat #013-12061) in DMEM-LG, 14nM BMP-2 (R&D Cat #355-BM-010) in 5 mM acetic acid and 0.1% bovine serumalbumine (BSA, Sigma Cat #A-8806); 1 M β-glycerophosphate (Sigma Cat#G9891) in Tyrode solution; Tyrode solution: 9.72 g Tyrode's salt (SigmaCat #T2145) and 1 g NaHCO3 in 1 L H₂O, ALP substrate buffer: 25 mMglycine and 0.5 mM MgCL2, pH 10.5; ALP substrate solution: 5 mgp-nitrophenyl phosphate (Sigma 104 substrate, Sigma Cat #50942-200TAB)in 3.75 ml ALP substrate buffer, pH 10.5; 1 mM p-nitrophenol (Sigma Cat#104-1) in ALP substrate solution.

Assay

For the ALP assays, MC3T3 1b cells were grown in assay culture medium(corresponding to the maintenance culture medium in the absence ofG418). MC3T3 1b were seeded in 200 μl at 3×10⁴ cells/ml if inductionstarted 72 h later or 2×10⁴ cells/nil if induction started 96 h later.The plates were incubated for 72 h or 96 h at 37° C. and 5% CO₂, beforethe induction was started using complete medium (by supplementing theculture medium with 10 mM b-glycero-phosphate (bGP) and 50 μM ascorbicacid (AA)). The antibodies to be tested were diluted with completemedium. The antibody together with BMP-2 (0.7 nM) and sclerostin (50 nM)were added to the wells (triplicate) in a final volume of 200 μlcomplete medium. On each plate, 4 internal controls were included intriplicate: a solvent control (BSA), BMP-2 control (0.7 nM),BMP-2+sclerostin (BMP-2 (0.7 nM) and sclerostin (50 nM)) and asclerostin control (50 nM). The plates were incubated for another 72 hat 37° C. and 5% CO₂. At the end of the induction period, the assay wasterminated by removing the medium and adding 150 μl ALP substratesolution (freshly prepared) to each well. The plates were incubated for3-30 min. One hundred μl of 1M NaOH was added to stop the reaction andthe plates were shaken on a Plateshaker. OD was red against a blank at405 nm and ALP activity was calculated in nmol/min. Fifty nM sclerostinwas used to get at least 70% inhibition of BMP-2 induced ALP production[0.7 nM BMP-2].

Wnt-Assay

This assay was established for testing antibodies based on the abilityof sclerostin to inhibit Wnt1-mediated induction of STF reporter gene.

Transfection of HEK293 Cells

On day 1, HEK293 cells were seeded at 1.3-1.4×10⁵ cells per well (in 0.5ml volume) of a 24-well poly-D-lysine plate (BD-BioCoat #356414) in DMEM(Gibco, Cat #61965-026) containing 10% fetal calf serum (FCS), 1%L-glutamine (Gibco, Cat #25030.024), 1% non essential amino acids(Gibco, cat #11140) without antibiotics. Transfection was performed onday 2 with Lipofectamine 2000 (Invitrogen, Cat #11668-019). For eachwell to be transfected, the following amount of plasmids was added to afinal volume of 50 μl OptiMEM®I (Gibco, Cat #31985-047): for controlwells (pcDNA3+, 480 ng; SuperTopFlash (STF) 20 ng; phRL-CMV, 0.5 ng) andfor Wnt1 treatment wells (pcDNA-wnt1, 20 ng; pcDNA3+, 460 ng;SuperTopFlash (STF) 20 ng; phRL-CMV, 0.5 ng).

In a second tube, 1.6 μl of Lipofectamine 2000 was diluted into 50 μl ofOptiMEM®I and incubated at room temperature for 5 minutes. The contentsof the two tubes were then mixed by adding the content of the lipid tubeto the DNA tube and incubated for 30 minutes at room temperature toallow DNA-lipid complex formation. The DNA-lipid complex (100 μl) wasthen evenly added to wells and incubated at 37° C. in 5% CO₂ for 5hours. At the end of the 5-hour incubation, cells were ready fortreatment with testing reagents such as SOST, antibodies etc.

Establish SOST-Dose Dependent Inhibition

To establish the dose inhibition curve, a series of two-fold dilutionsof rhSOST in DMEM containing 10% FCS, 1% L-glutamine and 1% nonessential amino acids without antibiotics were prepared, starting at 160nM. The concentration of stock rhSOST was 260 μg/ml in DMEM with 1% FCS.Routinely, each condition was tested in duplicates. Therefore, 1 ml ofmedium for each condition was prepared and 450 μl was added per wellafter removing medium containing transfection mix from wells. Thetreatment time was 18-20 hours. At the end of the incubation, luciferaseassay was performed as outlined below.

Test of Anti-SOST Antibodies

Antibodies were premixed with SOST before adding to the cells. For thispurpose, a DMEM medium (10% FCS, 1% L-glutamine and 1% non essentialamino acids without antibiotics) with 20 to 30 nM of rhSOST wasprepared. Then, different dilutions of tested antibodies were added tothe SOST containing medium according the experimental design. Thesemixes were prepared 40 minutes before the treatment. Each condition wasroutinely tested in duplicates. To do so, 1 ml of medium for eachcondition was prepared and 450 μl was added per well after removingmedium containing transfection mix from wells. The treatment time was18-20 hours. At the end of the incubation, luciferase assay wasperformed as outlined below.

Luciferase Assay

At the end of the incubation, medium was removed, and 300 μl of 1×Passive Lysis Buffer (Promega, Cat #E194A) was added to lyse cells.Luciferase activity was then measured using Dual-Glo Luciferase System(Promega, Cat #E2940) with 30 μl of lysates in duplicates. The assay wasperformed according to the instruction booklet provided with the kit.Typically, 30 μl of Dual-Glo luciferase (firefly luciferase; for STF)and 30 μl of Dual-Glo Stop and Glo (Renilla luciferase; for transfectionefficiency control) substrates were used. Luminescent signals weremeasured with Mithras LB940 instrument (Berthold Technologies).

Data Calculation

The ratio of firefly to Renilla luciferases was calculated. The finalresults are expressed by setting the value of Wnt1 without SOST as 1.

Mineralization Assay

Cells

MC3T3 1b cells is a clone of MC3T3-JP cells expressing OSE2-luc. Thisclone was obtained after stable transfection of MC3T3-JP (MC3T3-E1 mouseosteoblast, Japan clone, Jp; a kind gift of Dr. T. Kokkubo, Novartis,Japan) using 8x-OSE2wt-mOG2luc in pcDNA3.1+.

Culture Medium

MC3T3-1 b cells were routinely cultivated in minimum essential mediumalpha (MEMα; Invitrogen, Cat #22561-021) supplemented with 10% fetalcalf serum (FCS; Amimed Cat #2-01F100-I), 2 mM L-glutamine (Gibco Cat#25030-024), 50 IU penicillin/50 μg/ml streptomycin (Amimed Cat#4-01F00-H) and 10 mM Hepes (Gibco Cat#15630-056) and 0.75 mg/ml G418(Gibco Cat #10131-027) (maintenance culture medium).

Mineralization Assay

Matrix-associated calcium deposited in wells was determined in MC3T3-1 bcells using the Calcium kit (Axon Lab, Cat #AXON0012). Cells were seededat 6×10³ cells/well or 2×10³ cells/well to improve cell responsivenessin 96-well plates in 100 μl assay culture medium (maintenance culturemedium without G418) and incubated for 3 days to reach confluence. Assayculture medium was then changed and compounds to be tested addedtogether with 10 mM b-glycero-phosphate (bGP; Sigma Cat #G9891) and 50μM ascorbic acid (AA; Wako Pure Chemical Cat #013-12061). Prior to theiraddition to the cells, sclerostin and the Fabs to be tested werepre-incubated in a separate plate for 2 h at room temperature; meanwhilethe assay 96 well-plates were given 2.1 or 2.8 nM BMP-2 (R&D Systems,Cat #355-BM-010) before receiving the sclerostin-Fab mix. Cells wereincubated for 14 days and assay medium was replaced every 3-4 days.Briefly, at the end of the incubation, cells were washed twice with 200μl PBS/well, 50 μl of 0.5 M HCl was added in each well and plates werefrozen down at −20° C. for minimum 24 h. At the appropriate time, plateswere thawed at room temperature for 2 hours and 10 μl of each well wastransferred in a new 96-well plate. Calcium Working Solution (1:5) wasthen added (200 μl) and 5-30 minutes later, plates were read at 595 nmon a microplate reader.

The absorbance was translated into μg of calcium according to a standardcurve, control well (ascorbic acid and beta-glycerophosphate) value wassubtracted from each data well and final results were expressed as % ofBMP-2-induced mineralization.

Smad1 Phosphorylation Western

Material

MC3T3-E1 mouse osteoblast, (Japan clone, kind gift of Dr. T. Kokkubo,Novartis, Japan)

C3H10T1/2 cells (mouse embryo mesenchymal; ATCC, Cat. Nb.: CCL-226)

SCL non-glycosylated, E. coli derived (Novartis, PSU5257)

15N SCL non-glycosylated, E. coli derived (Novartis, PSU11274)

hSOST-APP, glycosylated, HEK-EBNA derived (Novartis, BTP11100)

rhSCL, glycosylated, mouse myeloma cells derived (R&D Systems, Cat. Nb.:1406-ST/CF)

anti hSOST antibody (R&D Systems, Cat. Nb.: AF1406)

PhosphoSafe Extraction Reagent (Novagen, Cat. Nb.: 71296-3)

Protease Inhibitor Cocktail Set III (Calbiochem, Cat. Nb.: 539134)

NuPAGE Novex Tris-Acetate Gel 7% 1.5 mm, 15 wells (Invitrogen, Cat. Nb.:EA03585)

Tris-Acetate SDS Running Buffer 20× (Invitrogen, Cat. Nb.: LA0041)

NuPAGE Antioxidant (Invitrogen, Cat. Nb.: NP0005)

Immobilon-P Transfer Membrane 0.45 um (Millipore, Cat. Nb.: IPVH00010)

Wattman chromatography paper (Merck, Cat. Nb.: 3587600)

XCell SureLock Mini-Cell and XCell II Blot Module (Invitrogen)

VersaMax microplate reader (Bucher)

Cell Culture and Extraction

MC3T3-E1 and C3H10T1/2 cells were routinely cultivated in minimumessential medium alpha (MEMα; Invitrogen, Cat #22561-021) or in DMEMwith high glucose (Invitrogen, Cat #41965-039), respectively. Allculture media were supplemented with 10% fetal calf serum (FCS;BioConcept Cat #2-01F10-I, lot. Z04459P), 2 mM L-glutamine (InvitrogenCat #25030-024), 50 IU penicillin/50 ug/ml streptomycin (Invitrogen Cat#15140-122) and 10 mM Hepes (Invitrogen Cat #15630-056) (maintenanceculture medium)

Cells were seeded in maintenance culture medium in 6 well plates (3ml/well) and grown until confluence (with 1.4×10⁵ MC3T3-1b cells/well,confluence was reached at day 3; with 1.0×10⁵ C3H10T1/2 cells/well,confluence was reached at day 3). Following overnight serum depletion inculture medium containing 1% FBS, the medium was replaced by fresh onesupplemented with 1% FBS, BMP-6 (R&D Systems, Cat #507-BP) and thesubstance(s) to be tested. Prior to their addition to the cells, BMP-6and the substance(s) to be tested were pre-incubated for 1 h at roomtemperature, as it had been described for a phospho-Smad 1/3/5 inC3H10T1/2 (Winkler, EMBO J., 2003, 22(23):6267-76). When testinganti-sclerostin antibodies, these were preincubated with sclerostinovernight at 4° C., before being incubated with 0.2 nM BMP-6 for 1 h atroom temperature and being finally added to confluent cells. After theappropriate treatment time, the cells were washed with 2 ml ice-coldPBS. One hundred μl/well PhosphoSafe Extraction Reagent and 1:200diluted Protease Inhibitor Cocktail were then added to the cells whichwere then incubated on ice for 5 min. Cells were scraped off the wellsand transferred into a microfuge tube. The cell extract was kept on icefor 15 min, interrupted by a vortex step every 5 min. Afterwards, thecell extract was centrifuged for 5 min at 16′000 g and 4° C. Finally,the supernatant was transferred into a fresh microfuge tube for proteindetermination.

Protein concentration in cell lysate was determined using the BCAProtein Assay Kit according to the manufacturer's instruction. BSA wasused as standard. For denaturation, the cell lysate was diluted 1:2 withLaemmli buffer (Bio-Rad, Cat #161-0737, containing β-Mercaptoethanol(1:20, Merck, Cat #1.12006) freshly added) and boiled for 5 min at 95°C. After cooling down, the samples were stored at −20° C. until furtheruse.

Western Blot

Smad (H-465) antibody (Santa Cruz, Cat. Nb.: sc-7153) is a rabbitpolyclonal IgG, raised against amino acids 1-465 representing fulllength Smad1 of human origin. It should recognize Smad1, Smad2, Smad3,Smad5 and Smad8 of human, rat and mouse origin. Phospho-Smad1(Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428) antibody (CellSignalling, Cat #9511) is a rabbit polyclonal IgG, raised against asynthetic phospho-peptide corresponding to residues surroundingSer463/465 of human mad5. It should detect endogenous levels of Smad1only when dually phosphorylated at serine 463 and serine 465, as well asSmad5 and Smad8 only when phosphorylated at the equivalent sites ofhuman, mouse, rat, mink and xenopous origin. The antibody does notcross-react with other Smad-related proteins.

The XCell SureLock Mini Cell system (Invitrogen) was prepared accordingto the manufacturer's instruction. The protein samples (2 μg for a Smad,5 μg for a Phospho-Smad Western analysis) were loaded in equal finalvolume on a 7% NuPAGE Novex Tris-Acetate Gel in 1× Running Buffer.SeeBlue Plus2 pre-stained standard (10 μl, 1:10; Invitrogen, Cat#LC5925) and MagicMark XP Western protein standard (10 μl, 1:100;(Invitrogen, Cat #; LC5602) were used as molecular weight markers. Thegel was run for 75 min with constant voltage (150 V).

Blotting pads and filter papers were soaked in 700 ml 1× NuPAGE TransferBuffer. A PVDF transfer membrane was first soaked for 30 sec in methanoland then transferred in 1× NuPAGE Transfer Buffer (Invitrogen, Cat#NP0006, Transfer Buffer:Methanol 10:1 freshly prepared). The gelcassette plates were separated with a gel knife, one pre-soaked filterpaper was placed on top of the gel and any trapped air bubbles carefullyremoved. The plate was turned up side down on Saran wrap and afterremoval of the plate, the pre-soaked transfer membrane was placed on thegel and any air bubbles were removed. A second pre-soaked filter paperwas placed on top and any air bubbles removed. Two soaked platting padswere placed into the cathode core of the Cell II Blot Module. Thegel/membrane sandwich was carefully picked up and placed on the blottingpads (with the gel closest to the cathode core). Finally, 3 pre-soakedblotting pads were placed on the membrane assembly and the anode corewas added on the top. The blot module was slid into the guide rails onthe lower buffer chamber and the wedge was locked into positionaccording to the manufacturer's instruction. The blot module was filledwith 1× NuPAGE Transfer Buffer until the gel/membrane assembly wascovered. The outer buffer chamber was filled with 650 ml deionized waterand the protein transfer to the PVDF membrane was performed withconstant voltage (30 V) for 2 hours.

Following the blotting, the membrane was first washed for 10 min in0.05% Tween 20 in PBS and then blocked under gentle shaking for 1 hourin 25 ml SuperBlock T20 Blocking Buffer at room temperature. The primaryantibody (Phospho-Smad 1/5/8 or Smad (H-465)) was added to the membranein a 1:1000 dilution in SuperBlock T20 Blocking Buffer (Pierce, Cat#37516) and the membrane was incubated overnight at 4° C. under shaking.The membrane was then washed 3 times for 10 min with 0.05% Tween 20(Fluka, Cat #93773) in PBS before being incubated with the RP conjugatedsecondary antibody (1:1000 in SuperBlock T20 Blocking Buffer) for 60 minat room temperature under shaking. A least 3 washing steps of 10 mineach were performed before the membrane was incubated for 5 min with theSuperSignal West Femto Substrate Working Solution (Pierce, Cat #34095).Finally, the membrane was placed in a plastic pocket and imaged withFluor-S Multilmager (Bio-Rad) and Camera. An optimal exposure time of 1to 2 min was determined by comparing images taken between 30 sec up to 5min.

Data Analysis

The chemiluminescence activities were measured using Quantity One(Bio-Rad) and the EC₅₀ values were calculated using XLfit4 software.Each phospho-Smad signal was normalized by its corresponding total Smadsignal.

LRP6/Sclerostin ELISA

Ninety six-well microtiter non-treated plates were coated with 100μl/well LRP6/Fc (1 μg/ml, R&D Systems, Cat #1505-LR) diluted in PBS. Ascontrol for non-specific binding (NSB), a few wells were filled with 100μl/well PBS. The plates were covered with plastic film and incubatedovernight at RT. Following the coating, plates were washed 3 times with200 μl/well 0.05% Tween 20 (Fluka, Cat #93773) in PBS and wells wereblocked for 1 h at 37° C. by adding 300 μl/well SuperBlock blockingbuffer (Pierce, Cat #37535) in TBS. After incubation, the block solutionwas removed and 100 μl/well sclerostin (E. coli derived, Novartis;1-1000 ng/ml) diluted in 1% BSA in PBS were added. The plates wereincubated for 2 h at RT before being washed 3 times with 200 μl/well0.05% Tween 20 in PBS. Afterwards, 100 μl/well anti sclerostin antibody(1 μg/ml) diluted in 1% BSA in PBS were added and plates incubated for 2h at RT before being washed 3 times with 200 μl/well 0.05% Tween 20 inPBS. Finally, 100 μl/well ALP conjugated anti Goat IgG Ab (1:5000; SigmaCat#A-7888) diluted in 1% BSA (Sigma Cat. Nb.: A-7888) in PBS were addedfor 1 h at RT and plates were then washed 3 times with 200 μl/well 0.05%Tween 20 in PBS. To determine ALP, 100 μl/well ALP substrate (Sigma, Cat#S0942) solution (1 tablet per 5 ml diethanolamine substrate buffer 1×;Pierce, Cat #34064) was added to the plates for 90 min and opticaldensity measured at 405 nm.

LRP4 Overexpression in Hek293 Cells

HEK293 cells (ATCC Cat #CRL-1573) were routinely cultivated in DMEM/F12(Invitrogen Cat #21331-020) supplemented with 10% FCS (BioConcept Cat#2-01F10-I, lot. Z04459P), 2 mM L-glutamine (Invitrogen Cat #25030-024),100 IU penicillin/100 μg/ml streptomycin (Invitrogen Cat #15140-122) and10 mM HEPES (Invitrogen Cat #15630-056). Hek293 cells were seeded at5×10⁴ cells/well in poly-D-Lysine 48 well-plate format and incubated for24 h before performing the transfection with Lipofectamine 2000(Invitrogen Cat #11668-019). For each well to be transfected, thefollowing amount if plasmids was added to a final volume of 25 μlOptiMEM®I (Gibco, Cat #31985-047) and mixed gently: for control wells(pmaxGFP, 62.5 ng, pcDNA3+, 125 ng; SuperTopFlash (STF) 62.5 ng;SV40-driving Renilla luciferase plasmid, 0.75 ng) and for Wnt1 treatmentwells (pmaxGFP, 62.5 ng, pcDNA-wnt1, 62.5 ng; pcDNA3+, 62.5 ng;SuperTopFlash (STF) 62.5 ng; SV40-driving Renilla luciferase plasmid,0.75 ng) and for LRP4-Wnt1 treatment wells (pcDNA3+-LRP4, 62.5 ng,pcDNA3+-wnt1, 62.5 ng; pcDNA3+, 62.5 ng; SuperTopFlash (STF) 62.5 ng;SV40-driving Renilla luciferase plasmid, 0.75 ng). In a second tube, 0.8μl of Lipofectamine 2000 was diluted to a final volume of 25 μl ofOptiMEM®I and incubated at room temperature for 5 minutes. The contentsin the two tubes were then mixed by adding the content in the lipid tubeto the DNA tube and incubated for 30 minutes at room temperature toallow DNA-lipid complex formation. The DNA-lipid complex (50 μl) wasthen evenly added to wells and incubated at 37° C. in 5% CO₂ for 5hours. At the end of the 5-hour incubation, cells were ready for a 20 htreatment with testing reagents such as SOST, DKK1 (R&D Cat #1096-DK),antibodies etc. The luciferase assay was performed as described under“Wnt-assay” but adding 150 μl Passive Lysis Buffer instead.

LRP4 Overexpression in C28A2 Cells

C28a2 cells (from Mary Goldring, Harvard Institutes of Medicine, Boston,Mass., US) were cultivated in the same medium as HEK293 (see above)except that HEPES was not present and a supplement of non-essentialamino acids (Gibco Cat #11140) was added. C28a2 cells were seeded at1×10⁵ cells/well in a 24-well plate format in antibiotic free culturemedium and incubated overnight before performing the transfection withLipofectamine 2000 (Invitrogen Cat #11668-019). For each well to betransfected, the following amount of plasmids (600 ng/well total) wasadded to a final volume of 50 μl OptiMEM®I (Gibco, Cat #31985-047) andmixed gently: for control wells (SuperTopFlash (STF) 100 ng;SV40-driving Renilla luciferase plasmid, 2 ng and pcDNA3+, 500 ng tocompensate) and for Wnt1 treatment wells (pcDNA-wnt1 plasmid, 100 ng;LRP5 plasmid, 100 ng; SuperTopFlash (STF) 100 ng; SV40-driving Renillaluciferase plasmid, 2 ng and pcDNA3+, 300 ng) and for LRP4-Wnt1treatment wells (pcDNA-wnt1 plasmid, 100 ng; LRP5 plasmid, 100 ng; LRP4plasmid, 100 ng; SuperTopFlash (STF) 100 ng SV40-driving Renillaluciferase plasmid, 2 ng and pcDNA3+, 200 ng). In a second tube, 1.6 μlof Lipofectamine 2000 was diluted into 48.4 μl of OptiMEM®I andincubated at room temperature for 5 minutes. The contents of the twotubes were then mixed by adding the content of the lipid tube to the DNAtube and incubated for 30 minutes at room temperature to allow DNA-lipidcomplex formation. The DNA-lipid complex (100 μl) was then evenly addedto wells and incubated at 37° C. in 5% CO₂ for 2 hours. At the end ofthe 2 hour incubation, transfection medium was replaced by 450 μl/wellantibiotic-free medium and cells were incubated for 24 h. Cells werethen treated for 20 h with testing reagents such as SOST, or DKK1 (R&DCat #1096-DK). The luciferase assay was performed as described under“Wnt-assay”.

LRP4 Knockdown Hek293 Cells

Wnt1/STF/Renilla Hek293 cells (a stable clone issued from the stabletransfection of Hek293 cells (ATCC Cat #CRL-1573)) with Wnt1 expressionplasmid, SuperTopFlash reporter plasmid, and a SV40-driving Renillaluciferase plasmid) were routinely cultivated in DMEM 4500 g/L glucose(Invitrogen Cat #41965-035) supplemented with 10% FCS (Amimed Cat#2-0F100-I), 2 mM L-glutamine (Invitrogen Cat #25030-081), 100 IU/mlpenicillin/100 μg/ml streptomycin (Gibco Cat #15140-163), 6 μg/mlpuromycin (Invitrogen Cat #ant-pr-1), 150 μg/ml zeocin (Invitrogen Cat#45-0430) and 150 μg/ml hygromycin (Invitrogen Cat #10687-010). Theselection antibiotics were left out during knockdown experiments. Cellswere seeded at 0.6×10⁵ cells/well in poly-D-Lysine 24 well-plate formatand left to attach overnight before performing the transfection of LRP4siRNAs with HiPerFect (Qiagen Cat #301707). The sense and anti-sensesequences of the LRP4 siRNA used were as follow:

(SEQ ID NOs: 160/161) LRP4a:TAAATTATCATAAAGTCCTAA/AGGACTTTATGATAATTTATT; (SEQ ID NOs: 162/163)LRP4b: ATAGTGGTTAAATAACTCCAG/GGAGTTATTTAACCACTATTT;(SEQ ID NOs: 164/165) LRP4c:TAAATTCTCGTGATGTGCCAT/GGCACATCACGAGAATTTATT; (SEQ ID NOs: 166/167)LRP4d: TTTCTTATAGCACAGCTGGTT/CCAGCTGTGCTATAAGAAATT;(SEQ ID NOs: 168/169) LRP4e:TAGACCTTTCCATCCACGCTG/GCGTGGATGGAAAGGTCTATT;For each well to be transfected, two eppendorf tubes were prepared: thefirst one contained 0.2 μl of a 20 nM stock of one of the LRP4 siRNA or0.1 μl of a 20 nM stock of two different LRP4 siRNA (the final totalconcentration in siRNA/well is 6.6 nM) and 50 μl Optimem and the secondone, 3 μl HiPerFect and 47 μl Optimem. The content of the second tubewas added to the first one, shortly vortexed and left for 10 minutes atroom temperature. One hundred μl of this mixture was then added to therespective well and, cells were incubated at 37° C. under 5% CO₂ for 30hours. Afterwards, transfection mixture was removed and replaced byfresh antibiotic free culture medium (450 μl/well) and left to incubatefor an other 24 hours. Before SOST or DKK1 treatment, medium wasremoved, replaced by fresh antibiotic free culture medium containingappropriate dilution of SOST or DKK1 (R&D Cat #1096-DK) and cells wereincubated for 20 hours at 37° C. under 5% CO₂. Luciferase determinationwas then performed as described under “Wnt-assay”.Animal Models

Eight-month-old female OF1/IC mice (n=16/group, Charles River, France)were administered twice weekly intravenously anti-sclerostin antibodyANTIBODY A (25 mg/kg, h/mlgG2a) (MOR05813) or control antibody(anti-PC-h/mlgG2a). Control groups received either daily subcutaneously100 microg/kg PTH(1-34) or PBS vehicle. Treatment lasted 2.5 weeks forall animals. Half of the animals (n=8/group) was sacrificed at that timepoint for histomorphometric analysis. Treatment continued for theremainder of the animals (n=8/group) up to 5 weeks.

Tibial bone mass and geometry of the animals was measured at theinitiation of treatment by peripheral quantitative computed tomography(pQCT) and micro computed tomography (microCT). Animals were distributedevenly according to body weight and tibial total bone mineral density asmeasured by pQCT into groups. Bone mineral density, mass and geometrychanges were evaluated after 2.5 and 5 weeks of treatment. Body weightwas monitored weekly. Animals which were sacrificed after 2.5 weeks oftreatment were administered two fluorochrome labels for marking of bonemineralization 10 and 3 days prior to necropsy. Blood was taken atnecropsy. Dual energy x-ray absorptiometry (DEXA) measurements werecarried out at necropsy on excised tibia, femur, and lumbar vertebrae.The bones were fixed, dehydrated, and embedded for microtome sectioningand histomorphometric analysis of bone formation dynamics.

Treatment Protocol

Control antibody: anti-PC-h/mlgG2a,

concentration: 2.5 mg/ml, application volume: 10 ml/kg

Vehicle: 50 mM Citrat, 140 mM NaCl

Anti-sclerostin antibody: anti-SOST-MOR05813, h/mlgG2a,

2.45 mg/ml, application volume: 10 ml/kg

Vehicle: 50 mM Citrat, 140 mM NaCl

hPTH (1-34) (Bachem, Bubendorf, Switzerland) 100 μg/kg

vehicle: PBS+BSA 0.1%

Treatment Groups:

1 isotype control iv.=anti-PC-mlgG2a

2 anti-SOST-MOR05813 iv.

3 vehicle control sc.=PBS+BSA 0.1%

4 hPTH(1-34) sc.

Maintenance Conditions

Animals were housed in groups of four to five animals at 25° C. with a12:12 h light-dark cycle. They were fed a standard laboratory dietcontaining 0.8% phosphorus and 0.75% calcium (NAFAG 3893.0.25, Kliba,Basel, Switzerland). Food and water were provided ad libitum.

Statement on Animal Welfare

Animal experimentation was carried out according to the regulationseffective in the Canton of Basel-City, Switzerland.

Methods

Peripheral Quantitative Computed Tomography (pQCT)

The animals were placed in a lateral position under inhalation narcosis(Isoflurane, 2.5%). The left leg was stretched and fixed in thisposition.

Cross-sectional bone mass, density and geometry was monitored in theproximal tibia metaphysis at level of the mid-fibula head and 1.8 mmdistal to the proximal end of the tibia as detected in the scout scanusing an adapted Stratec-Norland XCT-2000 fitted with an Oxford 50 AMX-ray tube and a collimator of 0.5 mm diameter. The following setup waschosen for the measurements: voxel size: 0.1×0.1×0.5 mm; scan speed:scout view 10 mm/s; final scan 3 mm/s, 1 block, contour mode 1, peelmode 2; cortical threshold: 610 mg/cm3, inner threshold: 610 mg/cm3.

Micro Computed Tomography (microCT)

The animals were placed in a lateral position under inhalation narcosis(Isoflurane, 2.5%). The left leg was stretched and fixed in thisposition.

Cancellous bone structure was evaluated in the left proximal tibiametaphysis using a Scanco vivaCT20 (Scanco Medical AG, Switzerland). Thenon-isometric voxels had a dimension of 10.5× 10.5×10.5 μm. From thecross-sectional images, the cancellous bone compartment was delineatedfrom cortical bone by tracing its contour. In all the other slices,boundaries were interpolated based on the tracing to define the volumeof interest. 143 slices within the area of the secondary spongiosa(starting below the lateral lower edges of the growth plate) wereevaluated. A threshold value of 370 was used for the three dimensionalevaluation of structural parameters.

Dual Energy X-Ray Absorptiometry (DEXA)

Ex vivo DEXA measurements were performed on the left tibia, the leftfemur and in the lumbar vertebrae 1-4. Ethanol (70%) was used for softtissue simulation. The measurements were performed using a regularHologic QDR-1000 instrument adapted for measurements of small animals. Acollimator with 0.9 cm diameter and ultrahigh resolution mode (linespacing 0.0254 cm, resolution 0.0127 cm) was used.

Fluorochrome Labelling:

Alizarin (20 mg/kg, subcutaneous, alizarin complexone, Merck, Dietikon,Switzerland) was a 10 days prior to necropsy

Calcein (30 mg/kg, subcutaneous, Fluka, Buchs, Switzerland)—3 days priorto necropsy

Histology and Histomorphometry

After dissection, the right femur and lumbar vertebrae five and six wereplaced for 24 h into Kamovsky's fixative, dehydrated in ethanol at 4°C., and embedded in resin (methylmethacrylate). Using a Microtome 2050Supercut (Reichert Jung, Arnsberg, Germany), a set of 5 μm-thicknon-consecutive microtome sections were cut in the frontal mid-bodyplane for evaluation of fluorochrome-label-based bone formation. Thesections were examined using a Leica DM microscope (Leica, Heerbrugg,Switzerland) fitted with a camera (SONY DXC-950P, Tokyo, Japan) andadapted Quantimet 600 software (Leica, Cambridge, United Kingdom). Onesection per animal was sampled. Microscopic images of the specimens weredigitalized and evaluated semi-automatically on screen. Bone perimeter,single and double-labelled bone perimeter, and interlabel width weremeasured (×200 magnification). Mineralized perimeter values (percent),mineral apposition rates (micrometers/day) (corrected for sectionobliquity in the cancellous bone compartment), and daily bone formationrate values (daily bone formation rate/bone perimeter [micrometers/day])were calculated. All parameters were evaluated in the secondaryspongiosa of the distal femur metaphysis and in one lumbar vertebra.Another set of sections were tartrate-resistant acid phosphatase (TRAP)stained. Osteoclast surface per bone surface (%) was evaluated in thesecondary spongiosa:

Statistical Analysis

Results are expressed as mean+/−SEM. Statistical analysis was carriedout using Student's t test (two-tailed; unpaired). Treatment(anti-sclerostin antibody or hPTH(1-34) was tested for difference tocontrol (control anti-body or PBS), *, +p<0.05, **, ++p<0.01.

Affinity Determination

Affinity Determination of Selected Anti-Human Sclerostin Fabs UsingSurface Plasmon Resonance (Biacore)

The kinetic constants k_(on) and k_(off) were determined with serialdilutions of the respective Fab binding to covalently immobilizedantigen sclerostin using the BIAcore 3000 instrument (BIAcore, Uppsala,Sweden). For covalent antigen immobilization standard EDC-NHS aminecoupling chemistry was used. Kinetic measurements were done in PBS (136mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.76 mM KH₂PO₄ pH 7.4) at a flowrate of 20 μl/min using Fab concentration range from 1.5-500 nM.Injection time for each concentration was 1 min, followed by 3 mindissociation phase. For regeneration 2×5 μl 10 mM glycine pH 1.5 wasused. All sensograms were fitted using BIA evaluation software 3.1(BIAcore).

Electrochemiluminescene (BioVeris) Based Binding Analysis for MeasuringAffinities of Sclerostin Binding Fab in Lysates

For the measurement of the affinity of sclerostin-binding antibodyfragments in E. coli lysates (BEL extracts), binding was analyzed by aBioVeris M-384 SERIES® Workstation (BioVeris Europe, Witney,Oxfordshire, UK).

The experiment was carried out in 96-well polypropylene microtiterplates and PBS supplemented with 0.5% BSA and 0.02% Tween 20 as assaybuffer. Biotinylated human sclerostin protein was immobilized on M-280Streptavidin paramagnetic beads (Dynal) according to the instructions ofthe supplier. 1:25 dilution of the bead stock solution was added perwell. 100 μl diluted BEL extract and beads were incubated overnight atRT on a shaker. For detection, anti-human (Fab)′2 (Dianova) labeled withBV-tag™ according to supplier instructions (BioVeris Europe, Witney,Oxfordshire, UK) was used.

Randomly picked clones were analyzed with the method described above.Clones giving the highest values were chosen for further analysis insolution equilibrium titration.

Determination of Affinities of Fabs to Sclerostin Using SolutionEquilibrium Titration (SET)

For KD determination, monomer fractions (at least 90% monomer content,analyzed by analytical SEC; Superdex75, Amersham Pharmacia) of Fab wereused.

Electrochemiluminescence (ECL) based affinity determination in solutionand data evaluation were basically performed as described by Haenel etal., 2005. A constant amount of Fab (25 pM) was equilibrated withdifferent concentrations (serial 3^(n) dilutions) of unlabelled human,mouse or Cynomolgus sclerostin (starting concentration: 500 pM) insolution. Biotinylated human sclerostin (0.5 μg/ml) coupled toparamagnetic beads M-280 Streptavidin, Dynal) and BV-tag™ (BioVerisEurope, Witney, Oxfordshire, UK) labeled anti-human (Fab)′2 antibody(Dianova) were added and incubated for 30 min. Subsequently, theconcentration of unbound Fab was quantified via ECL detection usingM-SERIES® 384 analyzer (BioVeris Europe).

Affinity improved Fab clones where identified by an ECL based highthroughput affinity screening BioVeris assay. After hit selection 4sub-clones were consolidated by the same method.

Receptor Binding Inhibition Potency Assay Using BioVeris™

For the BioVeris™-based binding inhibition potency assay recombinanthuman BMP-2 was directly coupled (NHS/EDC chemistry) to carboxylic acidM-270 magnetic beads (Dynal) according to supplier instructions. Theassay was performed in 96-well polypropylene microtiter plate (Nunc). 50μl/well of purified Fab in assay buffer (PBS+1% Tween 20 (stringent) or0.1% Tween 20 (less stringent)+1% BSA) were diluted in 1:3 dilutionsteps (staring concentration: 1000 nM). 50 μl/well of biotinylated humansclerostin (4 nM) was added to each Fab dilution. After incubation for90 min shaking at 400 rpm on an Eppendorf Thermomixer at 22° C., 25 μlof the BMP-2 coated beads (2.7E07 beads per ml) and 1:500 dilutedStreptavidin labeled with BV-tag™ according to supplier instructions(BioVeris Europe) were added to each well and incubated for 30 min (800rpm, 22° C.). Detection was performed by BioVeris M-384 SERIES®Workstation (BioVeris Europe). For EC₅₀ determination a 4-parameterlogistic fit model (XLfit, IDBS) was used.

Production of Immunoglobulins

Conversion Into the Human IgG1 Format and Human/Mouse IgG2a Format

In order to express full length IgG, variable domain fragments of heavy(VH) and light chains (VL) were subcloned from Fab expression vectorsinto appropriate pMorp®Ig vectors: pMorph®_h_Ig1 and chimerichuman/mouse pMorp®2_h/m_Ig2a. Restriction enzymes EcoRI, MfeI, BlpI wereused for subcloning of the VH domain fragment into pMorph®_h_IgG1 orpMorp®2_h/m_IgG2a and EcoRV, BsiWI, HpaI for subcloning of the VL domainfragment into pMorp®_h_Igκ, pMorp®_h_Igλ and pMorph®2_h/m_Igλ vectorsrespectively.

Restriction enzymes EcoRI, MfeI and BipI were used for subcloning of theVH domain fragment into pMORPH®_h_IgG1: the vector backbone wasgenerated by EcoRI/BlpI digestion and extraction of the 6400 by fragmentwhereas the VH fragment (350 bp) was produced by digestion with MfeI andBlpI and subsequent purification. Vector and insert were ligated viacompatible overhangs generated by the EcoRI and MfeI digests,respectively, and via the BlpI site. Thereby, both the EcoRI and theMfeI restriction site are destroyed.

Restriction enzymes MfeI and BlpI were used for subcloning of the VHdomain fragment into pMORPH®2_h/m_IgG2a. In this new generation of IgGvectors, upon other modifications, the EcoRI site (which allowed onlysub-cloning via compatible overhangs) was replaced by the MfeI site thusallowing MfeI/BlpI digestion of both, vector and insert.

Subcloning of the VL domain fragment into pMORPH®_h_Igκ was performedvia the EcoRV and BsiWI sites, whereas subcloning into pMORPH®_h_gλ andpMORPH®2_h/m_Igλ was done using EcoRV and HpaI.

Transient Expression and Purification of Human IgG

HEK293 or HKB11 cells were transfected with an equimolar amount of IgGheavy and light chain expression vectors. On days 4 or 5post-transfection the cell culture supernatant was harvested. Afteradjusting the pH of the supernatant to pH 8.0 and sterile filtration,the solution was subjected to standard protein A column chromatography(Poros 20A, PE Biosystems).

Example 1 Generation of Human and Cyno Recombinant Scierostin Proteins

The full-length cDNA coding for human sclerostin precursor (GenBank Acc.No AF326739) featuring a natural signal peptide (aa 1-213, NPL 005002)was cloned into the mammalian expression vector pRS5a by insertion intothe restriction sites Asp718 and Xba1. A protein detection andpurification tag (APP=EFRH) was added to the C-terminus of the gene.

Following small scale expression verification in transient 6-well-platetransfection assays, the generation of stable transfection pools wasinitiated by transfection of four pools (1.0×10E6 cells each) bylipofection. 48 hours post transfection selection of transfectants wasstarted by addition of the antibiotic Zeocin™ at 100 μg/mlconcentration. Once all four pools had resumed normal growth recombinantprotein titers were assessed by analytical affinity chromatography onanti-APP HPLC and the highest producing pool—Pool2—was selected foradaptation to serum-free medium and further scale-up. Simultaneously,large-scale transient expression trials on the 10-l-scale were alsoperformed using Polyethylenimine as carrier of plasmid DNA duringtransfection and the Wave™ bioreactor system (C20SPS-F, Art-No. 100.001,Wave Biotech) as culture system. The cell culture supernatants of 12-22l were harvested 7-10 days post transfection and concentrated bycross-flow filtration and diafiltration prior to purification.

Human and cyno SOST were purified by immunoaffinity chromatography.Briefly, batches of 10-20 L tissue culture supernatants wereconcentrated to 1-2 L by cross-flow filtration (cut off 10 kDa) andapplied at 2 ml/min on a 50 ml anti-APP Sepharose column prepared bycoupling the proprietary monoclonal anti-Ab1-40/APP (6E10-A5) to CNBractivated Sepharose 4B according to the manufacturer instructions (10 mgantibody per ml resin). After base-line washing with PBS, bound materialwas eluted with 100 mM glycine, pH 2.7 neutralized and sterile filtered.Protein concentration was determined by A280 using computed absorptionfactors. The purified proteins were finally characterized by SDS-PAGE,N-terminal sequencing and LC-MS. An aliquot of purified human SOST wasbiotinylated in PBS for 1 hour at 37° C. using 1 mM sulfo-NHS-lc-biotin(Uptima; UP54398A). Excess reagent was then removed by extensivedialysis against PBS.

E. Coli Derived Human SOST

His₆-PreScission tagged SOST aa24-213 (NPL006071, plasmid pX1504) andSOST aa24-213 (NPL006690, plasmid pXI515) were produced by refolding. E.coli Tuner (DE3) were transformed with either plasmid.

Batch PSU5257 was fermented on a 20 liter scale (V9405) using modifiedTB medium (version lab 112). Cells were induced at an OD600 of 3.90 with1 mM IPTG and induced for 3 hours 30 minutes at 37° C. Harvesting wascarried out using a continuous flow centrifuge, resulting in a wet cellpellet weighing 190 g.

Batch PSU11274 was fermented on a 20 liter scale in 16 L minimal M9-1medium supplemented with ¹⁵N-labelled ammonium chloride. Cells wereinduced with 1 mM IPTG once an OD600 of 1.5 had been reached andharvested at an OD600 of 3.3 using a continuous flow centrifuge,resulting in a wet cell pellet weighing 50 g.

Wet cell pellets from the above fermentation were lysed in 8 volumes of50 mM Tris pH 8.0 (containing 5 mM each EDTA, DTT and Benzamidine-HCl)using an Avestin C-50 emulsifier and centrifuged for 30 min at 12,000rpm. The supernatant was discarded and the resultant pellet re-suspendedin 10 volumes lysis buffer and centrifuged again. The process wasrepeated 3 more times, after which the lysis buffer was replaced withMilli-Q water, containing 5 mM DTT. The pellets were washed a furthertwo times under these conditions. Inclusion bodies were dissolved in 8MGuanidine-HCl (containing 100 mM DTT, 50 mM Tris pH 8 and 5 mM EDTA) for3-4 h at ambient temperature, then centrifuged at 20,000 rpm for 30 min,filtered through a 0.45 uM filter. Refolding was initiated byfast-dilution with 88 volumes (PSU11274) and 92 volumes (PSU5257)chilled refolding buffer (0.5M Tris containing 0.9M Arginine-HCl, 5 mMGSH and 0.5 mM GSSG pH 8). The solution was stored at 4° C. for 1 week.At this stage, the two preparations were treated slightly differently,because of the requirement to remove the his₆-tag from PSU5257 prior tocompleting refolding

PSU5257

The diluted folding solution was diafiltered against 1.5 volumes of 50mM Tris pH 8, 5 mM GSH, 0.5 mM GSSG by concentrating 2-fold, thendiluting back to the original volume. This was carried out 3 times,after which time, PreScission protease was added and the solution leftfor 48 h at 4° C. All concentrations/diafiltrations were carried outusing a Pellicon II ultrafiltration cassette with a 10 KDa cut-offmembrane. Finally the solution was concentrated 10-fold.

PSU11274

The diluted refolding solution was concentrated 10-fold. LC-MS was usedto confirm the formation of all 4 disulphide bridges prior toconcentration.

Both preparations were then dialysed against 2×10 volumes of 50 mMsodium acetate pH 5. Following successive filtration through glass wooland a 3.0 μm membrane to remove precipitated protein, purification wascarried out using SP-Sepharose™ high performance cation-exchangechromatography. The column was equilibrated with the dialysis buffer andsubsequently eluted with a 0-1M NaCl gradient in the same buffer over 20column volumes. Sclerostin eluted as a single peak with a slightshoulder, which was discarded. In the case of PSU5257, the main peak wascollected, concentrated subjected to size-exclusion chromatography usinga column of Superdex 75™, equilibrated with 50 mM Tris, 150 mM NaCl.With PSU11274, the sample was provided directly after cation-exchange.

Cynomolgus monkey SOST cDNA (cySOST) was amplified by RT-PCR withprimers based on the African green monkey SOST sequence (GenBankAccession #AF326742). The 5′ primer (ATGCAGCTCCCACTGGCCCTGTGTCTTGT) (SEQID NO: 170) corresponds to N-terminus of the signal peptide sequence andis not in the final secreted protein; the 3′ primer(AATCAGGCCGAGCTGGAGAACGCCTACTAG) (SEQ ID NO: 171) corresponds to aregion that is conserved among human, African green monkey and mouse.The amplified fragment was subcloned into the Bam HI/Eco RI sites ofpcDNA3.1(+) and sequence confirmed. This plasmid served further astemplate for a PCR amplification of cyno-SOST to add a C-terminal APPtag and attB recombination sites for final cloning into pDESTRS5aaccording to the Gateway technology [cySOST-pDESTRS5a].

Expression was done by large-scale transient transfection at the 10 Lscale in the Wave™ bioreactor system, harvested after 9 days posttransfection and purified as described above.

Example 2 Generation of Human Sclerostin-Specific Antibodies from theHuCAL GOLD® Library

Therapeutic antibodies against human sclerostin protein were generatedby selection of clones having high binding affinities, using as thesource of antibody variant proteins a commercially available phagedisplay library, the MorphoSys HuCAL GOLD® library.

HuCAL GOLD® library is a Fab library (Knappik et al., 2000) in which allsix CDRs are diversified by appropriate mutation, and which employs theCysDisplay™ technology for linking the Fab to the phage surface(WO01/05950, Löhning et al., 2001).

Selection by Panning of Sclerostin-Specific Antibodies from the Library

For the selection of antibodies recognizing human sclerostin severalpanning strategies were applied.

In summary, HuCAL GOLD® antibody-phages were divided into three poolscomprising different VH master genes.

These pools were individually subjected to either

a) a solid phase panning where the antigens (human and mouse sclerostin)were directly coated on Maxisorp 96 well microtiter plates (Nunc,Wiesbaden, Germany) or

b) a capture and semi-solution panning where the antigen (biotinylatedsclerostin), respectively the phage-antigen complex was captured on N/Aclear strips or

c) a solution pannings with biotinylated sclerostin where thephage-antigen complex was captured by Streptavidin magnetic beads(Dynabeads M-280; Dynal) for each panning pool.

Solid Phase Panning on Sclerostin

For the first round of the panning on sclerostin, wells of the Maxisorpplate were coated overnight with 300 μl (5 μg/ml) of human sclerostin(produced in HEK cells) diluted in PBS. After two washing steps, with400 μl PBS, the wells were incubated with blocking buffer containing 5%milk powder diluted in PBS.

Prior to the selections, HuCAL GOLD® phages were pre-adsorbed inblocking buffer (5% milk powder/PBS, 0.5% Tween 20) for 2 h at RT toavoid unspecific selection of antibodies.

After washing (2×400 μl PBS) of the coated and blocked Maxisorp plate,300 μl of the pre-adsorbed phages were added to the coated wells andincubated for 2 h at RT shaking gently. This incubation was followed by10 wash cycles with PBS and PBS/0.05% Tween 20 at RT.

Bound phages were eluted by adding 300 μl of 20 mM DTT in 10 mM Tris/HClpH 8.0 per well for 10 min at RT. The eluate was removed and added to 15ml E. coil TG1F⁺ cells grown to an OD_(600 nm) of 0.6-0.8. Phageinfection of E. coli was allowed for 45 min at 37° C. without shaking.After centrifugation for 5 min at 4120×g, the bacterial pellets wereeach re-suspended in 600 μl 2× YT medium, plated on LB-CG agar platesand incubated O/N at 30° C. Colonies were then scraped off from theplates and phages were rescued and amplified.

The second and third rounds of selection were performed in an identicalway to the first round of selection with the only difference that thewashing conditions after binding of phage were more stringent. In thesecond selection round for some panning conditions mouse sclerostin wasused as antigen in order to enrich for mouse cross-reactive antibodies.For some panning conditions another batch of human recombinantsclerostin (produced in E. coli) was coated.

Semi-Solution Panning on Sclerostin

Two different methods were performed for this kind of panning: a capturesemi-solution panning and the standard semi-solution panning procedure.

In detail, for capture semi-solution panning on biotinylated sclerostin,100 μl of the antigen was coated 2 h at RT on NeutrAvidin (N/A) clearstrips (from Pierce, activation level 100 μl, binding capacity 15 pmolper well). The N/A strips were blocked O/N at 4° C. with 200 μlChemiblock and washed twice with PBS.

The blocked phage solution (Chemiblock/0.05% Tween 20) was added toblocked N/A wells for 30 min and this step was repeated for another 30min in order to remove NeutrAvidin binders. Then the pre-cleared phageswere transferred to the biotininylated sclerostin immobilized on N/Aclear strips, sealed with foil and incubated O/N at RT shaking.

On the next day the phage solution was removed from the antigen coatedwells and the wells were washed.

For the standard semi-solution panning the blocked (Chemiblock/0.05%Tween 20), pre-cleared phages were added to a new pre-blocked 1.5 mlreaction tube (Chemiblock/0.05% Tween 20) and then biotinylated humansclerostin was added to a final concentration of 100 nM and incubatedO/N at RT shaking.

On the next day the phage-antigen solution from the 1.5 ml reaction tubewas added to new blocked wells of the NeutrAvidin strips and allowed tobind for 30 min at RT and then washed.

For both panning procedures bound phages were eluted after washing byadding 300 μl of 20 mM DTT in 10 mM Tris/HCl pH 8.0 per well for 10 minat RT. The eluate was removed and added to 15 ml E. coli TG1 cells grownto an OD_(600 nm) of 0.6-0.8. Phage infection of E. coil was allowed for45 min at 37° C. without shaking. After centrifugation for 5 min at4120×g, the bacterial pellets were each re-suspended in 600 μl 2× YTmedium, plated on LB-CG agar plates and incubated O/N at 30° C. Colonieswere then scraped off from the plates and phages were rescued andamplified.

The second and third rounds of selection were performed in an identicalway to the first round of selection with the only difference that thewashing conditions after binding of phage were more stringent.

Solution Panning on Sclerostin

For this type of panning 200 μl of Streptavidin magnetic beads(Dynabeads M-280; Dynal) were washed once with PBS and blocked withChemiblock for 2 h at RT. 500 μl of phages were blocked with Chemiblockfor 1 h at RT rotating. The blocked phages were twice pre-adsorbedagainst 50 μl blocked Streptavidin magnetic beads for 30 min. The phagesupernatant was transferred to a new blocked 2 ml reaction tube anddifferent concentrations of human biotinylated sclerostin were added(see Tables 5, 6 and 7) and incubated for 1 h at RT rotating. 100 μl ofthe blocked Streptavidin magnetic beads were added to each panning poolan incubated for 10 min on a rotator. The beads were collected with aparticle separator (Dynal MPC-E) for approx. 2.5 min and the solutionwas removed carefully.

After the wash cycles (Table 2), bound phages were eluted by adding 300μl of 20 mM DTT in 10 mM Tris/HCl pH 8.0 per well for 10 min at RT. Theeluate was removed and added to 15 ml E. coli TGIF⁺ cells grown to anOD_(600 nm) of 0.6-0.8. Phage infection of E. coli was allowed for 45min at 37° C. without shaking. After centrifugation for 5 min at 4120×g,the bacterial pellets were each re-suspended in 600 μl 2×YT medium,plated on LB-CG agar plates and incubated O/N at 30° C. Colonies werethen scraped off from the plates and phages were rescued and amplified.

The second and third rounds of selection were performed in an identicalway to the first round with the only difference that the washingconditions after binding of phage were more stringent. In the second andthird selection round for some panning conditions the antigenconcentration was reduced.

Subcloning and Expression of Selected Fab Fragments

Microexpression of Selected Fab Fragments

To facilitate rapid expression of soluble Fabs, the Fab-encoding insertsof the selected HuCAL GOLD® phages were subcloned via XbaI and EcoRIfrom the respective display vector into the E. coli expression vectorpMORPH®X9_MH (Rauchenberger et al., 2003). After transformation of theexpression plasmids into E. coli TG1 F⁻ cells chloramphenicol-resistantsingle clones were picked into the wells of a sterile 96-well microtiterplate pre-filled with 100 μl 2× YT-CG medium and grown O/N at 37° C. 5μl of each E. coli TG-1 culture was transferred to a fresh, sterile96-well microtiter plate pre-filled with 100 μl 2× YT mediumsupplemented with 34 μg/ml chloramphenicol and 0.1% glucose per well.The microtiter plates were incubated at 30° C. shaking at 400 rpm on amicroplate shaker until the cultures were slightly turbid (˜2-4 h) withan OD_(600 nm) of ˜0.5. To these expression plates, 20 μl 2× YT mediumsupplemented with 34 μg/ml chloramphenicol and 3 mM IPTG(isopropyl-β-D-thiogalactopyranoside) was added per well (finalconcentration: 0.5 mM IPTG), the microtiter plates were sealed with agas-permeable tape, and incubated overnight at 30° C. shaking at 400rpm.

Generation of whole cell lysates (BEL extracts): To each well of theexpression plates, 40 μl BEL buffer was added and incubated for 1 h at22° C. on a microtiter plate shaker (400 rpm).

Expression and Purification of HuCAL®-Fab Antibodies in E. Coli

Expression of Fab fragments encoded by pMORPH®X9_Fab_MH in E. coli TG1F-cells in larger scale was carried out in shaker flask cultures using750 ml of 2×YT medium supplemented with 34 μg/ml chloramphenicol.Cultures were shaken at 30° C. until the OD_(600 nm) reached 0.5. Fabexpression was induced by addition of 0.75 mM IPTG(isopropyl-β-D-thiogalactopyranoside) and cultivation for further 20 hat 30° C. Cells were harvested and disrupted using lysozyme and Fabfragments isolated onto Ni-NTA chromatography. The apparent molecularweights were determined by size exclusion chromatography (SEC) withcalibration standards. Concentrations were determined byUV-spectrophotometry (Krebs et al., 2001).

Example 2 Identification of Sclerostin-Specific HuCAL® Antibodies

Enzyme Linked Immunosorbent Assay (ELISA) for Detection of SclerostinBinding Fabs on Directly Coated Sclerostin

Maxisorp (Nunc, Rochester, N.Y., USA) 384 well plates were coated with20 μl of 2.5 μg/ml antigen (human sclerostin—produced in HEK cells,human sclerostin—produced in E. coli cells and mouse sclerostin) in PBS,pH 7.4 O/N at 4° C.

The plates were blocked with PBS/0.05% Tween 20 (PBST) containing 5%milk powder for 1 h at RT. After washing of the wells with PBST,BEL-extract, purified HuCAL GOLD® Fabs or control Fabs diluted in PBSwere added to the wells and incubated for 1 h at RT. To detect theprimary antibodies, the following secondary antibodies were applied:alkaline phosphatase (AP)-conjugated AffiniPure F(ab′)2 fragment, goatanti-human, anti-mouse IgG (Jackson ImmunoResearch). For the detectionof AP-conjugates fluorogenic substrates like AttoPhos (Roche) were usedaccording to the manufacturer's instructions. Between all incubationsteps, the wells of the microtiter plate were washed with PBST threetimes and five times after the final incubation with the secondaryantibody. Fluorescence was measured in a TECAN Spectrafluor platereader.

Capture Screening with Biotinylated Sclerostin

This kind of ELISA was used to screen for HuCAL GOLD® Fabs afterproceeding solution pannings.

Maxisorp (Nunc, Rochester, N.Y., USA) 384 well plates were coated with20 μl of 5 μg/ml sheep anti-human IgG, Fd fragment specific (The BindingSite, Birmingham, UK), diluted in PBS, pH 7.4.

After blocking with 3% BSA in TBS, 0.05% Tween 20 for 2 h at RT,periplasmic extracts or purified HuCAL GOLD® Fabs were added andincubated 1 h at RT. After washing the plates five times with PBST 20 μlof biotinylated sclerostin for specific binding or biotinylatedTransferin for unspecific binding were added to the wells.

Subsequently the biotinylated antigen sclerostin was allowed to bind tocaptured HuCAL®-Fab fragments. And after washing incubated withStreptavidin (Zymex) conjugated to alkaline phosphatase. For thedetection of AP-Streptavidin fluorogenic substrates like AttoPhos(Roche) were used according to the manufacturer's instructions (RocheDiagnostics, Mannheim, Germany). Fluorescence emission at 535 nm wasrecorded with excitation at 430 nm.

Results:

After the pannings the enriched phage pools were subcloned from thepMORPH® 23 library vector (allowing efficient antibody display on thephage surface) into the pMORPH® X9_Fab_MH expression vector whichmediates periplasmic expression of soluble Fabs. Single clones werepicked and soluble Fabs were expressed from these single clones.

In total, ˜4600 clones were analyzed in primary screening which wasperformed by binding of the Fabs directly from the bacterial lysates tohuman and mouse sclerostin immobilized on Maxisorp microtiter plates orby capture of Fabs via anti-Fd antibody to maxisorp microtiter platesfollowed by binding of biotinylated human sclerostin. Detection wascarried out in ELISA after labeling with an Alkaline Phosphatase-labeledanti-human (Fab)′₂ antibody or using Streptavidin alkaline phosphatase.

Hits obtained from the primary screening on recombinant sclerostin withsignals>5-fold over background were further analyzed for binding tohuman HEK and E. coli sclerostin and to mouse sclerostin either directlycoated or in solution using biotinylated sclerostin. Hits recognizingall sclerostin derivatives were selected and sequenced.

Characterization of HuCAL GOLD® Fabs

Affinity Determination Using Biacore

In order to further characterize the anti-sclerostin antibodies, theaffinity to human, mouse and Cynomolgus sclerostin was determined. Therecombinant sclerostin protein was immobilized on a CM5 Biacore chip andthe Fabs were applied in the mobile phase in different concentrations.For a reliable determination of monovalent affinities only such Fabbatches were used for Biacore measurements which showed ≧90% monomericfraction in a qualitative size exclusion chromatography.

Affinities for recombinant human, Cynomolgus and mouse sclerostin weredetermined in Biacore. Affinities are ranging from sub nanomolar to over500 nM on human, to nearly 5000 nM on Cynomolgus and to 4500 nM on mousesclerostin.

Example 3 Identification of Anti-Human Sclerostin Fab CandidatesInhibiting Sclerostin Binding to Immobilized BMP-2 Using the BioVeris™Device

All generated and purified Fabs were tested in the BioVeris-basedbinding inhibition potency assay for their ability to inhibit sclerostinbinding to recombinant human BMP-2. Two different stringency conditions(0.1% versus 1% Tween 20 in buffer system) were tested. 9 of 27 Fabswere able to inhibit sclerostin binding to immobilized BMP-2 understringent buffer conditions.

Example 4 Reversion of Sclerostin Inhibition of BMP-2 Induced ALPProduction in MC3T3 Cells

IgG Conversions of Parental Binders and Characterization of IgG

21 candidates were converted into the human IgG1 format by sub-cloninginto the pMORPH®_h_IgG1 expression vector and the corresponding lightchain constructs of the pMORPH®_h_Ig vector series, respectively.Expression was performed by transient transfection of HEK293 or HKB11cells and the full length immunoglobulins were purified from the cellculture supernatant. Functionality of IgG1s after purification wasassessed by ELISA binding to immobilized human, mouse and Cynomolgussclerostin.

IgGs in Primary Bio-Assay

All purified hlgGs were titered and tested in the ALP assay. The assaywas reliable with respect to BMP-2 sclerostin inhibition but theselected candidates could not show sclerostin inhibition in allexperiments (assay to assay activity variation, plate to platevariation, high variance within triplicate wells). Therefore only aranking of activity of the IgGs was possible.

Example 5 Affinity Maturation of Selected Anti-Sclerostin Fabs byParallel Exchange of LCDR3 and HCDR2 Cassettes

The results of testing the IgGs in the ALP assay allowed only a ranking.From this ranking, 4 Fabs were selected on high risk for affinitymaturation. All candidates have been selected to be optimized as singlelead candidates in L-CDR3 and H-CDR2.

To increase affinity and biological activity of the four selectedantibody fragments, LCDR3 and HCDR2 regions were optimized in parallelby cassette mutagenesis using directed mutagenesis (Virnekas et al.,1994), whereby the framework regions were kept constant. Prior tocloning of the maturation libraries, all parental Fab fragments weretransferred from the expression vector pMORPH® x9_MH into theCysDisplay™ maturation vector pMORPH® 25 via the XbaI/EcoRI restrictionsites. This vector provides the phage protein pill fused N-terminally toa cysteine residue as well as a C-terminal cysteine fused to the Fdantibody chain and thus allows disulfide-linked display of therespective Fab fragments on the phage surface.

For generation of the HCDR2 libraries the HCDR2 region of each parentalFab was excised and replaced by a 590 by stuffer. This DNA stufferfacilitates the separation of single digested from double digestedvector bands and reduces the background of the high-affinity parentalFabs during the maturation pannings. In a subsequent step, the stufferwas excised from the Fab-encoding plasmids of each parental clone andreplaced by the highly diversified HCDR2 maturation cassette.

In parallel, the LCDR3 region of five of the four parental clones wasreplaced by a diversified LCDR3 maturation cassette without intermediatecloning of a stuffer.

Sizes of the maturation libraries ranged between 1×10⁷ and 4×10⁸ cloneswith a cloning background below 1%, and quality control by sequencingrevealed a good quality of each library. Only the LCDR3 library ofparental clone MOR04520 had a low size (<10⁶) and many incorrect clonesand was therefore not included in the maturation pannings.

For each LCDR3 and HCDR2 maturation library, antibody-displaying phageswere prepared and phage titers determined by spot titration.

Panning Strategies for Affinity Maturation

The antibody-displaying phages from the following maturation librarieswere subjected to separate pannings and screenings:

Lead 1: MOR04518 (L-CDR3 maturation)

Lead 1: MOR04518 (H-CDR2 maturation)

Lead 2: MOR04520 (H-CDR2 maturation)

Lead 3: MOR04532 (L-CDR3 maturation)

Lead 3: MOR04532 (H-CDR2 maturation)

Lead 4: MOR04799 (L-CDR3 maturation)

Lead 4: MOR04799 (H-CDR2 maturation)

For each lead library three different pannings were performed. For eachpanning strategy different stringency conditions were applied. Toincrease panning stringency and to select for improved off-rates,extensive washing and competition with purified parental Fab or withsoluble recombinant sclerostin was performed. After the maturationpannings the enriched phagemid pools were sub-cloned into the pMORPH®x9_MH expression vector. About 2900 single clones were picked and Fabsexpressed by induction with IPTG.

BioVeris™ Affinity Ranking and Screening for Improved Affinities

For identification of affinity-improved sclerostin-specific Fabs thebacterial lysates of ˜2900 single clones were diluted and checked forFab binding to biotinylated human sclerostin immobilized onStreptavidin-coated beads. The binding was analyzed with a BioVerisWorkstation. Those clones giving the highest signals indicate improvedaffinity and were therefore chosen for further analysis by solutionequilibrium titration.

For this purpose, 176 single clones were selected and preliminaryaffinities were determined via 4-point solution equilibrium titration(SET) in BioVeris. From these data, 24 clones showing the bestaffinities were selected. These Fabs were purified in the mg scale.

Final affinities were determined using an 8-point SET measurement andhuman, mouse and cynomolgus sclerostin.

Optimized Fabs in Primary Bio-Assay

The optimized Fabs were tested in the cellular ALP assay. Most of themwere inactive; and variable reversal of sclerostin inhibition was seenwith some Fabs. Therefore a selection of clones was converted tohuman/mouse IgG2a format and tested in the same assay. But these clonesin the human/mouse IgG2a format could neither fully reverse sclerostininhibition and the wanted EC₅₀ (<10 nM) criterion could not be reached.The best matured candidate MOR05177 (VL matured from parental MOR04518)showed at 50 nM Fab or at 140-467 nM human/mouse IgG2a 75-85%restoration of the ALP signal.

Example 6 Identification of Anti-Sclerostin Antibodies from Parental andOptimized Fabs and IgGs in a New Primary Bio-Assay

Based on new publications (Wu et al. JBC 2005, He et al. JBC 2005,Bezooyen et al. ASBMR 2005, Winkler et al. JBC 2005), which show thatsclerostin impacts Wnt signaling directly or indirectly, a newfunctional bioassay was developed.

This assay is based on the ability of sclerostin to inhibitWnt1-mediated activation of STF in HEK293 cells.

In this assay all Fabs identified in the initial screenings plus allFabs identified in the affinity maturation were tested. It becameobvious the increased affinity of matured Fabs reflected in increasedpotency and efficacy in the wnt-1 assay.

Testing all parental Fabs identified two further antibodies as promisingtemplates for an additional affinity maturation. FIG. 1 shows theactivity of MOR05813_IgG2lambda, one of the most potent antibodyidentified in the wnt-1 assay.

Example 7 Characerization of Parental and Affinity Matured Fabs in OtherBio-Assays

Activity of Parental and Affinity Matured Fabs in Mineralization Assay

The mineralization assay is based on the ability of MC3T3 cells to forma mineralized matrix, indicating their capacity to undergo osteogenicdifferentiation. A strong mineralization (>1 μg calcium deposited perwell (96-well format)) was measured after 14 days at all BMP-2concentration tested. The addition of increasing sclerostinconcentrations induced a dose dependent inhibition of the BMP-2 (2.1 nM)induced mineralization (IC₅₀: 120 nM) (data not shown).

FIG. 2 shows an example of mineralization in the presence ofMOR05813_Fab. The antibody could restore BMP-2-induced mineralization tomaximum 80% of the initial response, whereas an anti-lysozyme Fab usedas negative control had no effect.

Activity of Parental and Affinity Matured Fabs in LRP6/Sclerostin ELISA

The LRP6/sclerostin ELISA is based on the ability of sclerostin to bindLRP6. To further characterize the Fabs produced, a selection of Fabs andIgGs were tested in this assay. In the presence of an anti-sclerostinantibody from R&D (1000 ng/ml=˜7 nM), sclerostin (0.9 nM) binding toLRP6 was inhibited by 68% compared to control (data not shown).

MOR05813_IgG2 lambda inhibited sclerostin binding to LRP6 up to 90% at90 nM, whereas an anti-lysozyme IgG used as negative control had noeffect on sclerostin binding to LRP6 (FIG. 3).

Activity of Parental and Affinity Matured Fabs in Phospho-Smad1 Assay

The phospho-Smad1 assay (Western) is based on the ability of BMP-6 toinduce Smad1 phosphorylation within 15 minutes in MC3T3-E1 cells.MOR05318_IgG2 lambda inhibited sclerostin action on BMP-6-induced Smad1phosphorylation with an EC₅₀ of 115 nM and restaured Smad1phosphorylation to maximum 66% of the BMP-6-induced phosphorylation. Thenegative control anti-lysozyme IgG (IgG C) had no effect (FIG. 4).

Example 8 Effect of LRP4, a Novel SOST-Interacting Partner, on theActivity of the Anti-SOST Antibody

With the exception of LRP4 siRNA e, LRP4 mRNA knockdown did not affectSTF activity in the absence of SOST. However, it did reduce the abilityof SOST to inhibit STF activity, between 10 and 30%. This was true forall five siRNA tested alone (FIG. 5A) or in different combinations (datanot shown). Upon overexpression LRP4 decreased SOST IC₅₀ by 5 and 16fold in the HEK and c28a2 supertopflash assay, respectively (FIGS. 5Band C). This effect was specific in the sense that overexpressed LRP4had no effect on DKK1 IC₅₀. Knockdown of LRP4 (siRNA) decreased theinhibitory action of SOST in Wnt-1 induced STF, whereas it did notdecrease the inhibitory action of DKK1 (FIG. 5D). In turn, LRP4decreased the action of the anti-SOST antibody by increasingMOR05813_IgG2a EC₅₀ from 17.2 nM to 30 nM (FIG. 5E). These data suggestthat LRP4 is a facilitator of SOST action.

Example 9 New Maturation

To increase affinity and biological activity of two newly selectedantibody fragments based on the results obtained from the bio-assays,LCDR3 and HCDR2 regions were optimized in parallel by cassettemutagenesis using directed mutagenesis (Virnekas et al., 1994), wherebythe framework regions were kept constant. Prior to cloning of thematuration libraries, all parental Fab fragments were transferred fromthe expression vector pMORPH® X9_MH into the CysDisplay™ maturationvector pMORPH® 25 via the XbalI/EcoRI restriction sites. This vectorprovides the phage protein pill fused N-terminally to a cysteine residueas well as a C-terminal cysteine fused to the Fd antibody chain and thusallows disulfide-linked display of the respective Fab fragments on thephage surface.

For generation of the HCDR2 libraries the HCDR2 region of each parentalFab was excised and replaced by a 590 by stuffer. This DNA stufferfacilitates the separation of single digested from double digestedvector bands and reduces the background of the high-affinity parentalFabs during the maturation pannings. In a subsequent step, the stufferwas excised from the Fab-encoding plasmids of each parental clone andreplaced by the highly diversified HCDR2 maturation cassette.

In parallel, the LCDR3 region of five of the four parental clones wasreplaced by a diversified LCDR3 maturation cassette without intermediatecloning of a stuffer.

Sizes of the maturation libraries ranged between 2×10⁷ and 2×10⁸ cloneswith a cloning background below 1%, and quality control by sequencingrevealed a good quality of each library. For each LCDR3 and HCDR2maturation library, antibody-displaying phages were prepared and phagetiters determined by spot titration.

Panning Strategies for Additional Affinity Maturation

The antibody-displaying phages from the following maturation librarieswere subjected to separate pannings and screenings:

Lead 1: MOR04525 (L-CDR3 maturation)

Lead 1: MOR04525 (H-CDR2 maturation)

Lead 2: MOR04529 (L-CDR3 maturation)

Lead 2: MOR04529 (H-CDR2 maturation)

For each lead or pool library three different pannings were performed,for each panning strategy different stringency conditions were applied.To increase the panning stringency and to select for improved off-rates,competition with soluble recombinant sclerostin protein was performedduring prolonged incubation and washing periods.

After the pannings the enriched phagemid pools were subcloned into thepMORPH® X9_MH expression vector. About 1600 single clones were pickedand Fabs expressed by induction with IPTG.

The affinity criterion of <100 pM for human sclerostin was fulfilled forderivatives of both parental Fabs. The must cross-reactivity withcynomolgus sclerostin and with mouse sclerostin of < 500 pM wasfulfilled for derivatives of both parental Fabs. MOR04525 yielded inmore clones having higher affinities to all three species.

Example 10 Characterization of Anti-Sclerostin Antibodies in In VivoStudies

Eight-month-old female OF1/IC mice (n=16/group, Charles River, France)were administered twice weekly intravenously anti-sclerostin antibodyMOR05813 (24.5 mg/kg, mlgG2a) or isotype control antibody(anti-PC-mlgG2a). Control groups received either daily subcutaneously100 microg/kg PTH(1-34) or vehicle (PBS+0.1% BSA). Treatment lasted 2.5weeks for all animals. Half of the animals (n=8/group) was sacrificed atthat time point for histomorphometric analysis. These animals hadreceived fluorochrome markers 10 and 3 days prior to necropsy forhistomorphometric evaluation of bone formation dynamics. Treatmentcontinued for the remainder of the animals (n=8/group) up to 5 weeks.

The animals were monitored for changes in bone mass, density andgeometry. In vivo peripheral quantitative computed tomography [pQCT]demonstrated that MOR05177 is strongly bone anabolic in the proximaltibia of aged mice increasing bone mineral content (FIG. 6) and density(FIG. 7). The bone anabolic effect occurred both in the cortical (FIG.8) and cancellous (FIG. 9) bone compartment. These data suggest that theobserved inhibitory effect of MOR05813 on sclerostin action in the Wntsignaling reporter assay in non-osteoblastic cells translates intoinduction of bone formation responses due to sclerostin inhibition invivo. The magnitude of the bone anabolic response in mice was comparableto bone anabolism induced by a high dose of hPTH(1-34).

MicroCT analysis demonstrated increases in trabecular bone volume (FIG.9) mainly related to a thickening of trabecular bone structuresconsistent with bone anabolism (FIG. 10).

Bone mineral density increased further in animals, which were treated upto 5 weeks (FIG. 11). Bone mineral density as evaluated ex vivo by DEXAwas increased in the appendicular (tibia, femur) and axial (lumbarvertebrae) skeleton (FIG. 12-14). The effect was comparable to the onemeasured in the positive control group treated daily with 100 microg/kghPTH(1-34).

Histomorphometric fluorochrome marker based analyses of bone formationdynamics demonstrated that the bone mass gain was due to a substantialincrease in bone formation rates in the appendicular (FIG. 15) and axialskeleton (FIG. 18). The effects were comparable to those of a high doseof PTH(1-34). Increases in bone formation rates were both related toincreases in mineral appostion rates (FIG. 16) and mineralizing surface(FIG. 17). Bone resorption was not increased by the treatment asdemonstrated by osteoclast surface measurement (FIG. 19).

Example 11 Screening Antibodies that Cross-Block Sclerostin BindingAntibodies of the Present Invention

Biacore Cross-Blocking Assay

The following generally describes a suitable Biacore assay fordetermining whether an antibody or other binding agent cross-blocks oris capable of cross-blocking antibodies according to the invention. Itwill be appreciated that the assay can be used with any of thesclerostin binding agents described herein.

The Biacore machine (for example the BIAcore 3000) is operated in linewith the manufacturer's recommendations.

Sclerostin may be coupled to e.g. a CM5 Biacore chip by way of routinelyused amine coupling chemistry, e.g. EDC-NHS amine coupling, to create asclerostin-coated surface. In order to obtain measurable levels ofbinding, typically 200-800 resonance units of sclerostin may be coupledto the chip (this amount gives measurable levels of binding and is atthe same time readily saturable by the concentrations of test reagentbeing used).

An alternative way of attaching sclerostin to the BIAcore chip is byusing a “tagged” version of sclerostin, for example N-terminal orC-terminal His-tagged Sclerostin. In this format, an anti-His antibodywould be coupled to the Biacore chip and then the His-tagged sclerostinwould be passed over the surface of the chip and captured by theanti-His antibody.

The two antibodies to be assessed for their ability to cross-block eachother are mixed in a stochiometrical amount, e.g. at a one to one molarratio, of binding sites in a suitable buffer to create the test mixture.The buffer used is typically a buffer which is normally used in proteinchemistry, such as e.g. PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄,1.76 mM KH₂PO₄, pH 7.4). When calculating the concentrations on abinding site-basis the molecular weight of an antibody is assumed to bethe total molecular weight of the antibody divided by the number oftarget (i.e. sclerostin) binding sites on that antibody.

The concentration of each antibody in the test mixture should be highenough to ensure saturation of the binding sites for that antibody onthe sclerostin molecules which are bound on the BIAcore chip. Theantibodies in the mixture are at the same molar concentration (on abinding basis) and that concentration would typically be between 1.0 mMand 1.5 mM (on a binding site basis).

Separate solutions containing the separate antibodies on their own arealso prepared. The buffer used for these separate solutions should bethe same buffer and at the same concentration as was used for the testmixture.

The test mixture is passed over the sclerostin-coated BIAcore chip andthe binding recorded. The bound antibodies are thereafter removed bytreating the chip with e.g. an acid, such as 30 mM HCl for about 1minute. It is important that the sclerostin molecules which are bound tothe chip are not damaged.

The solution of the first antibody alone is then passed over thesclerostin-coated surface and the binding is recorded. Thereafter, thechip is treated to remove all of the bound antibody without damaging thechip-bound sclerostin, e.g. by way of above mentioned acid treatment.

The solution of the second antibody alone is then passed over thesclerostin-coated surface and the amount of binding recorded.

The maximal theoretical binding can be defined as the sum of the bindingto sclerostin of each antibody separately. This is then compared to theactual binding of the mixture of antibodies measured. If the actualbinding is lower than that of the theoretical binding, the twoantibodies are cross-blocking each other.

ELISA-Based Cross-Blocking Assay

Cross-blocking of an anti-sclerostin antibody or another sclerostinbinding agent may also be detected by using an ELISA assay.

The general principle of the ELISA-assay involves coating ananti-sclerostin antibody onto the wells of an ELISA plate. An excessamount of a second, potentially cross-blocking, anti-sclerostin antibodyis then added in solution (i.e. not bound to the ELISA plate). A limitedamount of sclerostin is then added to the wells.

The antibody which was coated onto the wells and the antibody insolution will compete for binding of the limited number of sclerostinmolecules. The plate is then washed to remove sclerostin that has notbound to the coated antibody and to also remove the second, solutionphase, antibody as well as any complexes formed between the second,solution phase antibody and sclerostin. The amount of bound sclerostinis then measured using an appropriate sclerostin detection reagent. Anantibody in solution that is able to cross-block the coated antibodywill be able to cause a decrease in the number of sclerostin moleculesthat the coated antibody can bind relative to the number of sclerostinmolecules that the coated antibody can bind in the absence of thesecond, solution phase, antibody.

This assay is described in more detail further below for two antibodiestermed Ab-X and Ab-Y. In the instance where Ab-X is chosen to be theimmobilized antibody, it is coated onto the wells of the ELISA plate,after which the plates are blocked with a suitable blocking solution tominimize non-specific binding of reagents that are subsequently added.An excess amount of Ab-Y is then added to the ELISA plate such that themoles of Ab-Y sclerostin binding sites per well are at least 10 foldhigher than the moles of Ab-X sclerostin binding sites that were used,per well, during the coating of the ELISA plate. Sclerostin is thenadded such that the moles of sclerostin added per well are at least25-fold lower than the moles of Ab-X sclerostin binding sites that wereused for coating each well. Following a suitable incubation period, theELISA plate is washed and a sclerostin detection reagent is added tomeasure the amount of sclerostin specifically bound by the coatedanti-sclerostin antibody (in this case Ab-X). The background signal forthe assay is defined as the signal obtained in wells with the coatedantibody (in this case Ab-X), second solution phase antibody (in thiscase Ab-Y), sclerostin buffer only (i.e. no sclerostin) and sclerostindetection reagents. The positive control signal for the assay is definedas the signal obtained in wells with the coated antibody (in this caseAb-X), second solution phase antibody buffer only (i.e. no secondsolution phase antibody), sclerostin and sclerostin detection reagents.The ELISA assay needs to be run in such a manner so as to have thepositive control signal be at least 6 times the background signal.

To avoid any artifacts (e.g. significantly different affinities betweenAb-X and Ab-Y for sclerostin) resulting from the choice of whichantibody to use as the coating antibody and which to use as the second(competitor) antibody, the cross-blocking assay needs to be run in twoformats: 1) format 1 is where Ab-X is the antibody that is coated ontothe ELISA plate and Ab-Y is the competitor antibody that is in solutionand 2) format 2 is where Ab-Y is the antibody that is coated onto theELISA plate and Ab-X is the competitor antibody that is in solution.

Example 12 ELISA to Detect the Effect of MOR05813_IgG2Lambda on SOSTBinding of LRP6

LRP6/Sclerostin ELISA

Ninety six-well microtiter non-treated plates were coated with 100μl/well LRP6/Fc (1 μg/ml, R&D Systems, Cat #1505-LR) diluted in PBS. Ascontrol for non-specific binding (NSB), a few wells were filled with 100μl/well PBS. The plates were covered with plastic film and incubatedovernight at RT. Following the coating, plates were washed 3 times with200 μl/well 0.05% Tween 20 (Fluka, Cat #93773) in PBS and wells wereblocked for 1 h at 37° C. by adding 300 μl/well SuperBlock blockingbuffer (Pierce, Cat #37535) in TBS. After incubation, the block solutionwas removed and 100 μl/well sclerostin (E. coli derived, Novartis;1-1000 ng/ml) diluted in 1% BSA in PBS were added. The plates wereincubated for 2 h at RT before being washed 3 times with 200 μl/well0.05% Tween 20 in PBS. Afterwards, 100 μl/well anti sclerostin antibody(1 μg/ml) diluted in 1% BSA in PBS were added and plates incubated for 2h at RT before being washed 3 times with 200 μl/well 0.05% Tween 20 inPBS. Finally, 100 μl/well ALP conjugated anti Goat IgG Ab (1:5000; SigmaCat #A-7888) diluted in 1% BSA (Sigma Cat. Nb.: A-7888) in PBS wereadded for 1 h at RT and plates were then washed 3 times with 200 μl/well0.05% Tween 20 in PBS. To determine ALP, 100 μl/well ALP substrate(Sigma, Cat #S0942) solution (1 tablet per 5 ml diethanolamine substratebuffer 1×; Pierce, Cat #34064) was added to the plates for 90 min andoptical density measured at 405 nm.

Activity of Parental and Affinity Matured Fabs in LRP6/Sclerostin ELISA

The LRP6/sclerostin ELISA is based on the ability of sclerostin to bindLRP6. To further characterize the Fabs produced, a selection of Fabs andIgGs were tested in this assay. In the presence of an anti-sclerostinantibody from R&D (1000 ng/ml=˜7 nM), sclerostin (0.9 nM) binding toLRP6 was inhibited by 68% compared to control (data not shown).MOR05813_IgG2 lambda inhibited sclerostin binding to LRP6 up to 90% at90 nM, whereas an anti-lysozyme IgG used as negative control had noeffect on sclerostin binding to LRP6 (FIG. 20).

Example 13 Co-Treatment Using MOR05813

MOR05813+Zoledronic Acid

Eight-month-old female OF1/IC mice (n=10/group, Charles River, France)were ovariectomized to induce bone loss by estrogen deprival or leftintact. The animals were administered twice weekly intravenouslyanti-sclerostin antibody MOR05813 (24 mg/kg, h/mlgG2a) or controlantibody (anti-PC-h/mlgG2a, intact and OVX control groups). Additionalgroups received either a single application of zoledronic acid alone(100 μg/kg) or in combination with the anti-sclerostin antibodyMOR05813. Antibody treatment lasted 3.5 weeks (7 applications).

Tibial bone mass and geometry of the animals was measured prior toovariectomy by peripheral quantitative computed tomography (pQCT).Animals were distributed evenly according to body weight and tibialtotal bone mineral density into groups. Bone mineral density, mass andgeometry changes were evaluated at the end of the treatment period.

Results are expressed as mean+/−SEM. Statistical analysis was carriedout using RS1 (series 1999 for Windows, Domain Manufacturing Corp.,USA). The data were subjected to one-way analysis of variance (ANOVA).Equality of variances was tested by Levene F-test and differencesbetween groups using the Bonferroni-adjusted Dunnett test. Treatedgroups were tested for significance of differences from the controlantibody treated OVX group (p<0.05*, p<0.01**).

Ovariectomy induced bone loss can be blocked by the antibody treatmentin aged mice (FIG. 21). When the antibody is used in combination with asingle intravenous injection of the bisphosphonate zoledronic acid, boneloss is blocked and bone gain is induced as indicated by increases intotal bone mineral content (FIG. 21A) and density (FIG. 21B) as well asincreases in cortical thickness (FIG. 21C) and cancellous bone mineraldensity (FIG. 21D).

MOR05813+Alendronate Pre-Treatment

4.5-month-old female OF1/IC mice (n=10/group, Charles River, France)were administered twice weekly intravenously anti-sclerostin antibodyMOR05813 (10 mg/kg, h/mlgG2a) or control antibody (anti-PC-mlgG2a,intact and OVX control groups). Additional groups had received 7 weeksalendronate pre-treatment (4 μg/kg/day; 5 days/week) before receivingeither the control antibody or anti-sclerostin antibody MOR05813.Antibody treatment lasted 3.5 weeks (7 applications).

Tibial bone mass and geometry of the animals was measured prior toinitiation of antibody treatment by peripheral quantitative computedtomography (pQCT). Animals were distributed evenly according to bodyweight and tibial total bone mineral density into groups. Bone mineraldensity, mass and geometry changes were evaluated at the end of thetreatment period.

Results are expressed as mean+/−SEM. Statistical analysis was carriedout using RS1 (series 1999 for Windows, Domain Manufacturing Corp.,USA). The data were subjected to one-way analysis of variance (ANOVA).Equality of variances was tested by Levene F-test and differencesbetween groups using the Bonferroni-adjusted Dunnett test. Alendronatepre-treated groups were tested for significance of difference from thegroups receiving no pretreatment (p<0.05*, p<0.01**).

Long-term pretreatment with the bisphosphonate alendronate does not havenegative impact on the bone anabolic action of the anti-sclerostinMOR05813 as reflected by increases in total bone mineral content (FIG.22A) and density (FIG. 22B) as well as increases in cortical thickness(FIG. 22C) and cancellous bone mineral density (FIG. 22D). Due to thepersisting anti-resorptive properties of the bisphosphonate beyond theadministration period, an increase is observed in total bone mineralcontent (FIG. 22A) and cortical thickness (FIG. 22C).

MOR05813+DKK1 or hPTH

Six-month-old female nude mice (n=8/group) were administered twiceweekly intravenously vehicle, anti-sclerostin antibody MOR05813 (10, 20and 40 mg/kg, IgG2), anti-Dkk1 antibody (10 mg/kg, IgG1), hPTH(1-34)(100 microg/kg) or combinations thereof. Antibody treatment lasted 4weeks (8 applications).

Tibial bone mass and geometry of the animals was measured prior totreatment by peripheral quantitative computed tomography (pQCT). Animalswere distributed evenly according to body weight and tibial total bonemineral density into groups. Bone mineral density, mass and geometrychanges were evaluated at the end of the treatment period.

Results are expressed as mean+/−SEM. Statistical analysis was carriedout using RS1 (series 1999 for Windows, Domain Manufacturing Corp.,USA). The data were subjected to one-way analysis of variance (ANOVA).Equality of variances was tested by Levene F-test and differencesbetween groups using the Bonferroni-adjusted Dunnett test. Groups weretested for significance of difference from the vehicle treated group(p<0.05*, p<0.01**).

Bone anabolic effects increase with dose of anti-sclerostin MOR05813(FIG. 23). Co-treatment with an anti-Dkk1 antibody results in animproved increase in total bone mineral content (FIG. 23A) and density(FIG. 23B) and cortical thickness (FIG. 23C) and a synergistic increasein cancellous bone mineral density (FIG. 23D). Co-treatment withhPTH(1-34) results in a synergistic increase in all measured parameters(FIG. 23A-D).

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1. An isolated antibody or antigen binding portion thereof that binds toa sclerostin polypeptide, comprising: (a) the heavy chain variableregion (VH) polypeptide amino acid sequence as set forth as SEQ IDNO:70; (b) the light chain variable region (VL) polypeptide amino acidsequence as set forth as SEQ ID NO:81; or (c) the VH polypeptide aminoacid sequence as set forth as SEQ ID NO:70 and the VL polypeptide aminoacid sequence as set forth as SEQ ID NO:81.
 2. The antibody or antigenbinding portion according to claim 1, wherein said antibody or antigenbinding portion: a) binds to said sclerostin polypeptide with a K_(D)less than 1 nM; b) blocks the inhibitory effect of sclerostin in a cellbased Wnt signalling assay; c) has an IC₅₀ less than 100 nM as measuredin a cell-based Wnt signalling assay in HEK293 cell lines in thepresence of sclerostin; d) blocks the inhibitory effect of sclerostin ina cell based mineralization assay; e) has an IC₅₀ less than 500 nM asmeasured in BMP2-induced mineralization assay in MC3T3 cells in thepresence of sclerostin; f) inhibits LRP6/sclerostin interaction in asolution inhibition assay; g) has an IC₅₀ less than 10 nM as measured inLRP6/sclerostin ELISA h) blocks the inhibitory effect of sclerostin onBMP6 induced Smad1 phosphorylation in a cell-based functional assay; ori) has an IC₅₀ less than 500 nM as measured in BMP6 Smad1phosphorylation assay in a MC3T3-E1 cell line in the presence ofsclerostin.
 3. The antibody or antigen binding portion according toclaim 1, comprising a full length heavy chain amino acid sequence havingat least 95 percent sequence identity to the amino acid sequence setforth as SEQ ID NO:114.
 4. The antibody or antigen binding portionaccording to claim 1, comprising a full length light chain amino acidsequence having at least 95 percent sequence identity to the amino acidsequence set forth as SEQ ID NO:125.
 5. An isolated antibody or antigenbinding portion thereof that binds to a human sclerostin polypeptide,comprising: a) a VH which comprises, in sequence, (i) a CDR1 comprisingan amino acid sequence consisting of SEQ ID NO:4; (ii) a CDR2 comprisingan amino acid sequence consisting of SEQ ID NO:15; and (iii) a CDR3comprising an amino acid sequence consisting of SEQ ID NO:26; and b) aVL which comprises, in sequence, (i) a CDR1 comprising an amino acidsequence consisting of SEQ ID NO:37; (ii) a CDR2 comprising an aminoacid sequence consisting of SEQ ID NO:48; and (iii) a CDR3 comprising anamino acid sequence consisting of SEQ ID NO:59.
 6. The antibody orantigen binding portion according to claim claim 1 or claim 5,comprising the VH polypeptide amino acid sequence as set forth as SEQ IDNO:70 and the VL polypeptide amino acid sequence as set forth as SEQ IDNO:81.
 7. The antibody or antigen binding portion according to claim 6,which is an antibody, wherein said antibody is a human antibody or achimeric antibody.
 8. A pharmaceutical composition comprising theantibody or antigen binding portion according to claim 1 or claim
 5. 9.The pharmaceutical composition of claim 8, in combination with one ormore of a pharmaceutically acceptable excipient, diluent or carrier. 10.The pharmaceutical composition of claim 9, comprising an additionalactive ingredient.
 11. An isolated polynucleotide sequence encoding theantibody or antigen binding portion of claim 1 or claim
 5. 12. A cloningor expression vector comprising one or more polynucleotide sequences ofclaim
 11. 13. A host cell comprising the vector according to claim 12.14. A process for the production of an antibody or antigen bindingportion thereof, comprising: (a) culturing a host cell comprising acloning or expression vector comprising a polynucleotide encoding theantibody or antigen binding portion of claim 1 or claim 5, underconditions (i) wherein said vector expresses said polynucleotide; and(ii) wherein said polynucleotide is translated to said antibody orantigen binding portion; and (b) isolating said antibody or antigenbinding portion.
 15. A diagnostic kit comprising the antibody or antigenbinding portion according to claim 1 or claim
 5. 16. A method foridentifying a cell or tissue expressing sclerostin, the methodcomprising contacting said cell or tissue with the antibody or antigenbinding portion of claim 1 or claim 5, wherein said antibody or antigenbinding portion further comprises a detectable label.
 17. The methodaccording to claim 16, wherein said label is radioactive, fluorescent,magnetic, paramagnetic, or chemiluminescent.
 18. A method for increasingbone formation, comprising administering to a patient in need thereof atherapeutically effective amount of an antibody or antigen bindingportion as set forth in claim 1 or claim
 5. 19. The method according toclaim 18, wherein said patient is suffering from a disease or disorderselected from the group consisting of primary and secondaryosteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta,avascular necrosis, fracture healing, implant healing, and bone loss.20. A method for increasing bone mass, bone mineralization or bonedensity, comprising administering to a patient in need thereof atherapeutically effective amount of an antibody or antigen bindingportion as set forth in claim 1 or claim
 5. 21. The method according toclaim 20, wherein said patient is suffering from a disease or disorderselected from the group consisting of primary and secondaryosteoporosis, osteopenia, osteomalacia, osteogenesis imperfecta,avascular necrosis, fracture healing, implant healing, and bone loss.22. The method according to claim 21, wherein said bone loss is due toHIV infection, cancer, or arthritis.
 23. The method according to claim20, wherein said antibody or antigen binding portion thereof is anantibody comprising the VH polypeptide amino acid sequence set forth asSEQ ID NO:70 and the VL polypeptide amino acid sequence set forth as SEQID NO:81.
 24. The method according to claim 23, wherein said antibody isa human antibody or a chimeric antibody.
 25. The method according toclaim 20, further comprising administering an additional agent selectedfrom the group consisting of a bisphosphonate, a parathyroid hormone, aparathyroid hormone releasing agent, alendronate, an LRP4 neutralizingantibody and a DKK-1 neutralizing antibody.
 26. The method according toclaim 25, wherein said additional agent is a bisphosphonate, and whereinsaid bisphosphonate is zoledronic acid.
 27. The method according toclaim 25, wherein said additional agent is a parathyroid hormone, andwherein said a parathyroid hormone is hPTH(1-34).